aims of nucleic acid transfection

Table of contents :

• oligonucleotides
• gene fragments
• genes (transgene => transgenic organism)
• whole loci (translocus)
• chromosome-derived natural fragments (transchromosome => trans-chromosomic organism)
• transfection ways / gene transfer agents (GTA) : an ideal vector must (those marked in purple are useful also in vivo) ...
• have flexible tropisms
• be capable of extended circulation in the bloodstream (for intravenous administration)

Long circulation times allow the vector to passively accumulate in the tumour through a phenomenon known as the enhanced permeability and retention (EPR) effect or passive targeting. This phenomenon is due to the increased permeability of tumour vasculature that permits large macromolecules to extravasate into the tumour tissue, where they are retained due to the high hydrostatic pressure in the tumour. The biodistribution and elimination patterns of macromolecular systems are dictated by :
• particle size :
• particles with Ø > 5-7 mm are cleared by capillary filtration mainly in the lungs
• particles with Ø < 5 mm are generally cleared from the circulation by cells of the RES in organs such as liver and spleen.
Particle Ø may increase in the plasma due to aspecific interations.
• surface charge (zeta potential) :
• negatively charged particles are taken up by Kupffer cells by scavenger RME.
• positively charged particles accumulate in the lungs (except for cationic liposomes containing stearylamine or aminoglycolipids, which accumulate in liver and spleen due to its large surface area permitting adhesion of the cationic particles with the negatively charged cell surfaces).
• neutral particles exhibit an increased circulation half-life.
• other physicochemical properties that govern how the particles will interact with
• blood cells
• serum proteins (including serum nucleases)
• endothelium
• be small enough to gain access to :
• tissues (extravasation through fenestrated endothelium)
• cells (diffusion)
• cytosol (escaping endosome-lysysome processing after endocytosis)
• nucleus (entry nuclear pores)
• allow gene transcription
Host immune response to vector may be decreased by ...
• ... decreasing host immunoreactivity :
• immunosuppressive chemotherapies
• immune tolerance
• oral tolerance by feeding the host with UV inactivated vector
• gene therapy by co-expression of tolerogenic mAbs
• sequential administration method (vector first and DNA then)
• ... decreasing vector immunogenicity :
• masking of the capsid components of the virus by covalently linking hydrophilic moieties ("stealth" vectors)
• polyethylene glycol (PEG)
• N-(2-hydroxypropyl)methacrylamide (HPMA)
• microencapsulating the vector in biodegradable microparticles
• sodium alginate-based biodegradable microparticles
• decreasing the inflammatory properties of unmethylated CpG motifs in bacterial DNA coming from cloning procedures :
• methylation of CpG sequences
• reduction of the CpG frequency by ...
• eliminating non-essential regions with PCR amplification
• site-directed mutagenesis
• use of specific inhibitors of the CpG-TLR9 signalling pathway, such as chloroquine or quinacrine
• adding genes coding for inhibitors of the MHC class I presentation pathway.
• removing genes coding for immunogenic proteins (described below for each vector)
• sequential administration of different serotypes of the same viruses or of orthologous viruses (e.g. human adenovirus (HAd), bovine adenovirus (BAd) and porcine adenovirus (PAd))
• retargeting of viral vectors to specific cell receptors other than natural one(s)
Available vectors
• vectors allowing nucleic acid maintainance in all daughter cells
• chromosome integrating, replication-defective viral vectors : non-essential genes are provided in trans by an helper virus, a plasmid or in the genome of the packaging cell line / producer cell line / vector-producing cells (VPC)
• Retroviridae
• Gammaretrovirus : they have no NLSs, so their genome can be integrated only after nuclear envelope breakdown during M phase, which occurs only in turning-over cells, but not in permanent cells (this can be an advantage when targeting tumor cells !) : anyway also some kinds of stable cells can be induced to enter cell cycle (e.g. hepatocytes). v-oncogenes are removed.
• Moloney murine leukaemia virus (MoMLV / MMLV / MuLV / MLV) : gag, pol and env genes are replaced by neo^R, SV40 and transgene to create a SAX (that conserves 5' LTR, y, PB and 3' LTR).

MMLV integrates upstream or downstream the start site of transcriptionally active genes. Amphotropic MLV was originally isolated from feral mice and found to replicate in cells from various mammalian species, including humans. The env gene of isolate 4070A was subsequently used for transduction of primate cells with the newly developed MLV vectors. As a routine procedure the FDA recommends testing samples of the cell banks, vector supernatant and ex vivo transduced cells by infection on a Mus dunni fibroblast line (permissive also to the 75-bp viral enhancer elements-deficient and CMV promoter-recombinant amphotropic strains) and subsequent detection in a PG-4 S+L-indicator assay.
Capacity : 7.5 Kbp.
Max [particles] > 10⁸ / mL.
Rare preexisting immunity and no adaptive immune response elicited in humans.
In vivo expression : usually killed by C1 and anti-a-Gal Abs in serum => used in vivo only in immunoprivileged sites or ex vivo.
• Lentivirus : unlike other retroviruses, lentivectors have NLSs and do not necessarily require cell division for proviral integration and productive infection, so their genome can integrate even in non-dividing cells. Lentivirus typically insert into host DNA as a single non-rearranged copy. It is unknown how many integration events yield the desired level of transgene expression. However, up to 20 insertions can be detected in any one cell. Integration is known to trigger DNA repair mechanisms. Retroviral infection can also result in the generation of non-productive, "dead-end" circular molecules. These may, in themselves, be toxic to the cell.

Production method : typically producer cell lines are transfected with
• 3 plasmids method
• vector plasmids, containing the transgene, lentiviral LTRs for host cell integration and perhaps the Rev-responsive element (RRE) for most efficient vector production
• helper plasmids 1, encoding the gag and pol viral structural genes, in order to supply RT and integration functions for the therapeutic vector particles
• helper plasmids 2, encoding envelope proteins for the therapeutic viral particles and perhaps Rev protein. Difficulties in targeting specific cell types can be bypassed by modifying the envelope proteins to change tropism (pseudotyping) :
• amphotropic envelope : receptor is Pit2 / Ram1 (expressed on both apical and basolateral surfaces of human airway epithelial cells : anyway factors other than apical receptor abundance and the glycocalix inhibit gene transfer in the apical surface; low expression in HSCs).
• xenotropic envelope : receptor is XPR1.
• VSV-G envelope glycoprotein : receptors are several glycosaminoglycans (GAGs) : heparin (heparan sulfate) and dextran sulfate > desulfated heparin > chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate)
• 2 plasmids method : genetic elements have been inserted into a single kind of helper plasmids to transcriptionally partition structural and envelope helper components.
None of the starter plasmids are, by themselves, capable of functioning as autonomous lentiviruses : only the therapeutic vector contains the packaging signal and thus, in theory, infectious particles should contain only the envisaged transgene. In addition, most accessory genes have been removed during the process.
The contamination of vector supernatants with plasmid DNA results in a functional titer (ie the concentration of vector particles capable of transducing a target cell) which can be estimated by ...
• protein assays : these assesments do not distinguish between full (viral RNA genome-containing) and empty particles.
• immunoassays based on Gag (for example, p24 Gag for HIV-1, where 1 pg of p24 Gag corresponds to 12,000 physical particles)
• Pol (product-enhanced reverse transcriptase (PERT) assays)
• quantitative immunostaining methods
• nucleic acid assays
• assessing vector RNA in supernatant (RNA titer) : it is technically the easiest and provides the most rapid turnaround. Unfortunately, a RNA titer may not accurately reflect the functional titer due to presence of defective interfering (DI) particles, inhibitors of transduction, and carry over of plasmid DNA from packaging cell lines during vector production, leading to an overestimation of functional titer.
• assessing expression of a transgene following transduction : it is technically simple. The limitations of this method are two-fold : it assumes that the level of expression of all integrated vectors is above the detection threshold of the assay and may not distinguish cells with multiple copies of the vector, leading to an underestimation of functional titer.
• assessing vector DNA integration in transduced cells (DNA titer) : the most rigorous (it doesn't rely on vector expression and will detect multiple vector integration) but also the most time-consuming, requiring real-time quantitative PCR on lentiviral genomes.
Max [particles] > 10⁸ / mL. Lentiviral preparations can be concentrated by binding viral particles to an anion exchange column (anion exchange chromatography): viral particles are eluted with sodium chloride, desalted and further concentrated by ultrafiltration.
Viruses :
• nonprimate lentiviruses
• Equine lentiviruses
• Equine infectious anemia virus (EIAV)
• Feline lentiviruses
• Feline immunodeficiency virus (FIV)
• Primate lentivirus group
• Simian immunodeficiency virus (SIV)
• HIV-1. It appears that HIV-1 preferentially integrates into the human genome at the location of active genes (anywhere in the transcriptional unit but not upstream of the transcriptional start), genome regions with increased gene density, cytogenetic light bands, and GC-rich regions. The data point to unexpectedly strong biases in integration site selection, with regional hotspots including a 2.4 kb region containing 1% of sites
• Spumaviruses
• Human foamy virus (HFV)
• Type C retroviruses
• Murine sarcoma virus
• Myeloproliferative sarcoma virus (MPSV)
• PCC4-cell-passaged MPSV (PCMV)
• Murine embryonic stem cell virus (MESV) is a mutant expressed in EC but not in ES with host range properties expanded to embryonic cell lines other than ES and EC cells (e.g. fibroblasts)
• Spleen focus-forming virus (SFFV) / mink cell focus-inducing (MCF) is a replication-defective retrovirus in the Friend virus complex
Friend-MCF/MESV (FMEV) hybrid vector has been developed by the combination of the SFFVp U3 with the leader sequences from MESV.
Down-regulation of retroviral vector expression occurs in a number of cell types : although a number of vector elements have been shown to affect expression in specific experimental situations, the results can vary depending on the specific cDNA being expressed, the individual retroviral elements included in vectors, the promoter, or the inclusion of selectable markers. Deletion of all the transcriptional regulatory elements from the vector LTR, inclusion of permissive primer binding site (PBS) sequence, and use of a eukaryotic housekeeping promoter could greatly increase the number of expressing cell and the level of expression.
Strategies : as retrovirus replication genes are transactivated by NFkB, all STP which lead to NFkB activation may trigger retrovirus replication and so infection of bystander cells. E.g. T lymphocytes can be used as vector producing cells (VPCs) when they are co-transfected with a constitutively expressed TAA-specific TcR and the whole genome of a retroviral vector. The STP which follows TcR binding to the TAA on the neoplastic cell causes NFkB activation and allows formation of retrovirions : the recombinant retrovirus in turn codes for a transgene whose expression is under the control of the same TF that allows expression of the TAA, so that another control level is ensured.
Risks :
• while it seems wise to use the same vector preparation in both patients and pre-clinical animal studies, vectors with human gene promoters may behave significantly differently in animal hosts.
• because lentivectors are manufactured in eukaryotic cells, the level of contamination of the viral gene envelope with producer cell membrane molecules should be considered.
• generation of replication competent retrovirus (RCV) (including replication competent lentiviruses (RCL)) ...
• during vector production due to recombination of vector plasmids. Likelihood can be reduced by :
• removal of sequence homology between split-component systems plasmids
• codon usage
The net result of such engineering is that multiple recombination events would be required to produce infectious RCL. In theory, plasmids with gag, pol or VSV-G env could be packaged into viral particles. Cross-packaging of the therapeutic vector by other retroviruses and of other retroviruses by the vector should occur at demonstrably low levels.
Conditional packaging systems are worth exploring :
• dependence on synthetic tRNA
• packaging constructs using a heterologous promoter only capable of function in producer cell lines
Self-inactivating (SIN) vectors are deleted within the 3' LTR. This allows SIN lentivectors to productively infect and integrate into target cell populations, but generation of proviral transcripts is blocked. However, SIN vectors are difficult to manufacture. 3 RNA features have been identified that elevate retroviral transgene expression: an intron in the 5' untranslated region (5'UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3'UTR. To include such elements into SIN vectors with potentially improved safety, the strong retroviral promoter is excised from the U3 region of the 3' LTR and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5'UTR, taking advantage of retroviral splice sites surrounding Psi. Although titers of SIN vectors are about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allows production of > 10⁶ infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors show greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely match or even exceed the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells)^ref.
• in vivo due to mobilization of the vector proviral DNA by
• endogenous retroviruses in the genomes of patients. Most endogenous retroviral sequences in the human genome have large deletions and so cannot produce infectious virus, but the possibility that retroviral function could be supplied in trans or that the vector genome could recombine with human endogenous oncoretroviral sequences merits consideration.
• infectious retroviruses, such as during parallel infection with HIV-1 or HIV-2
Experience from other retroviral vectors studies suggests that any RCLs could be preducted from triple recombination product between vector, packaging plasmid, and env-containing plasmids. As VSV-G confers broad host cell range, carry-over of VSV-G env DNA or mRNA sequences into vector preparations should be minimized by cogent manufacturing and downstream purification strategies. It is worth nothing that the presence of a high concentration of vector material can cause interference in the detection of RCLs. Because all RCLs will have Gag/Pol (most likely, system-derived), measurement of polymerase activity, such as product-enhanced reverse transcriptase (PERT) assays, are to be encouraged. Aliquots from the viral lot can be chemically disrupted and a standard template for RT supplied to the solution. In the presence of Pol activity, cDNA is generated and a PCR reaction amplifies this sequence. These assays can be highly sensitive, where the detection of 10-100 Pol-positive particles is possible. However, it is undesirable to generate a lentivirus encoding VSV-G and Gag/Pol simply to calibrate these assays or to monitor RCL infectivity assays, since such a "positive control" virus could itself be pathogenic. However, alternative approaches should be taken to validate the sensitivity of these assays. The probability of recombination after transient transfection with 3 plasmids is much higher than the probability of recombination in stable producer cell lines containing single-copy cassettes with integrated elements. This is so provided that superinfection is not occurring to a significant extent in the long-term culture of the producer cells, as can happen using the VSV-G envelope. Standard tests for RCL in vector preparations involve a variety of in vitro infectivity-type assays, usually employing some tissue culture amplification procedures followed by measurement of parameters indicative of RCL propagation
• physical
• biochemical
• biological
• p24 Gag measurements
• enzyme marker rescue
• rescue of cell proliferation
• quantitative PCR techniques assuming that appropriate primers are available
These are opportunities for RCL generation in vivo, but any RCL that arises will have an Env different from the VSV-G of lentivectors. For example, if the vector genome is mobilized by HIV-1, it will have HIV-1 Env. Such RCLs only infect human cells and therefore there can be difficulties in establishing appropriate animal model systems. Immunodeficient rodents, such as SCID-hu mice, engrafted with human haematolymphoid cells, may be suitable for checking the transmissability of RCLs in vivo. Finally, patients should be monitored for vector mobilization. It is worth remembering that the long-term persistence of proviral DNA could result in RCL generation at any point in the patient's future.
• insertional mutagenesis due to lentivector proviral DNA integration, leading to ...
• silencing
• apoptosis
• oncogenesis
• lymphomas have occurred from administration of high titres of RCRs to immunosuppressed monkeys
• in humans, HIV-1 integration may be responsible for some lymphomas
• murine bone marrow cells transduced with a MMLV-based replication-deficient oncoretroviral vector containing a transgene encoding a truncated form of low-affinity NGFR (dLNGFR) caused leukemia by integration int the ecotropic viral integration site 1 (Evi1) gene. However, preclinical rodent studies may not provide reliable preductions on th insertional mutagenesis/oncogenesis issue for clinical administrations of lentivectors or lentivector-transduced cells, as the number of events required for transformation could be less than in humans, perhaps due to differences in telomerase regulation. In some haemotological malignancies, a relatively minor number of mutagenic events, perhaps as low as 3, is required for complete transformation; for other cancers, up to 8 may be necessary.
• see under X-SCID
Although numerous alterations of the coding and cis-acting sequences have been made in 3rd-generation lentivectors, a central component in all of these systems is the LTR. This may itself contribute to the oncogenic potential of some retroviruses. The second genetic change to develop cancer usually comes from the therapeutic protooncogene itself.
• germline alteration resulting in transgenerational effects
While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome^ref. HIV-1, ASLV, and MLV showed highly favored and disfavored bases as far as 20 bases upstream and 17 bases downstream, with some bases appearing at frequencies up to 2-fold higher or lower than expected. Among the three viruses, the patterns of base preferences differed both in the specific bases and the sites at which an integration occurred. The researchers found that the preferences of HIV-1 and ASLV had a strong axis of symmetry corresponding to virus-specific integration spacing, whereas MLV showed no such symmetry. This dissimilarity may represent fundamental differences in the way retroviral integration complexes interact with host DNA^ref.
The consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK) were analyzed. Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that 20% of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo, with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo. Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation^ref.
Integration-deficient lentiviral vectors can mediate efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. Substantial rescue of clinically relevant rodent models of retinal degeneration has been shown. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis^ref.
Cdkn2a^-/- mice are susceptible to a broad range of cancer-triggering genetic lesions. HSCs from these tumor-prone mice to assess the oncogenicity of prototypical retroviral and lentiviral vectors. HSCs were transduced in matched clinically relevant conditions, and compared integration site selection and tumor development in transplanted mice. Retroviral vectors triggered dose-dependent acceleration of tumor onset contingent on long terminal repeat activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by lentiviral vectors and did not enrich for specific integrants, despite the higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages^ref
• dissemination of new viruses from gene therapy patients
Hybrid vectors incorporating the integration ability of retroviruses (packaging components or whole genome) with the high titre, broad host-range of Ad vectors can overcome the respective disadvantages of either vectors :
• MLV + first generation of Ad vectors
• controllable integration into the genome
• transposons : reconstitution of an ancient transposon, Sleeping Beauty, from sequence alignment of nonfunctional remnants of members in the Tc1/mariner superfamily of transposons within the genomes of salmonids, provided the first functional transposon for use in vertebrate species. Sleeping Beauty has been used to accomplish stable chromosomal integration of functioning genes in somatic cells of adult mice.
• several phage integrases and their corresponding recognition elements
• contemporaneous administration of 2 vectors : the first one codes for the transgene flanked by transposons and the second one for a transposase (e.g. phiC31 bacteriophage integrase, which stably integrates large DNA sequences containing a specific 285-bp attB sequence into genomic "pseudo-attP sites". This approach may be further enhanced by directed evolution of integrases toward even more selective targeting of site-specific genomic integration.
• replicating episomal vectors (REV) can contain inserts in excess of 300 kb. They are based on viral origins of replication from dsDNA viruses :
• HHV-4 / EBV : EBV-based plasmid vectors are known to self-replicate in cells and carry EBNA1 gene and the oriP element. The EBNA1 protein binds to oriP, and facilitates the replication of the plasmid in synchrony with chromosomal DNA. Furthermore the EBNA1 also facilitates nuclear localization of the plasmid DNA.
• BK polyomavirus (BKV)
• Bovine papillomavirus (BPV) type 1 : transforming genes E5, E6 and E7 are not required whereas E1 and E2 are essential and sufficient for BPV-1 replication, episomal maintenance at intermediate to high copy number and stable, high-level expression of gene products.
• SV40
Combinations of viral and mammalian chromosomal origins of replication have also been found to improve nuclear retention of the episomes. Nucleosome assembly on REVs is more organised and resembles native chromatin, whereas nucleosome assembly on non-replicating, transiently transfected plasmids is less well ordered allowing greater access of transcription factors to target sequences. These observations imply that transcriptional control elements with a potent chromatin remodelling capacity (e.g. LCRs) may be required for efficient long-term tissue-specific gene expression from within REVs.
• vectors not allowing nucleic acid maintainance in daughter cells : this strategy is effective only for permanent cells and rapid cell killing. Otherwise repeated treatment are necessary => risk of strong immune responses and toxicity.
• fusion of the target cell with a cell that already has the transgene : fusion may be induced by Parainfluenzavirus 1 (Sendai virus) or polyethylen glycole (PEG)
• uncomplexed ("naked") plasmid DNA (pDNA) in saline solution^ref (see also plasmid DNA purification : current good manufacturing practice (cGMP) conditions for obtaining clinical-grade plasmid DNA) administered via ...
• injection
• intranuclear microinjection (1 to 1 efficiency => used mainly for germinal cell transfection)
• cytosolic microinjection (1 to 1 efficiency => used mainly for germinal cell transfection)
• into a vessel : owing to rapid degradation by nucleases in the serum and clearance by the mononuclear phagocyte system, the expression level and the area after injection of naked DNA are generally limited. Transient stop of blood flow downstream and upstream can allow stable binding of DNA with the receptor and internalization into cells.
• into a tissue
• intramuscular injection
• intradermic injection (usually more effective than intramuscular injection)
• implantable matrices made up of biodegradable polymers for sustained release of genes (it augments local gene transfer).
• ex vivo : solids that are formed outside the body to precise dimensions (in a range of shapes and sizes : from microcapsules and microspheres to rods, films, and 3D matrices), and then inserted into the body for gene delivery
• in vivo : the biodegradable implant forms in situ upon injection
• water-insoluble biodegradable polymer dissolved in a pharmaceutically acceptable water-miscible solvent (glycofurol). Upon injection the water-miscible solvent diffuses away from the polymer solution and the polymer coagulates or precipitates to form a solid polymeric implant. The DNA is encapsulated within the polymer matrix as it solidifies. After solidification, the DNA is then released by similar mechanisms as those for the solid pre-formed implants. During the solidification process, there is a process of convection and diffusion of glycofurol out of the polymer matrix. This mechanism is probably responsible for the accumulation of DNA at the edges of the matrix as the polymer coagulates, as well as for part of the initial fast release before solidification of the matrix. The DNA distribution in the center of the implant appears to be uniform as indicated by the absence of large aggregates. Interconnecting channels or compartments containing DNA are formed inside the implants : nevertheless, other channels do not appear to be connected, suggesting that the DNA entrapped within them would be released only after degradation of the polymeric matrix.
• thermosensitive polymers can control the release of encapsulated DNA in response to temperature changes that lead to swelling or de-swelling of the hydrated polymer
• poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA)
• biodegradable thermosensitive polymers
• PEG-poly(D,L-lactic acid-co-glycolic acid (PLGA)-PEG triblock copolymer. It can be loaded with plasmid DNA in aqueous phase at 4-20°C (room temperature) : at above 30-33°C (eg, at the body temperature), the solution-to-gel transition occurs and the DNA can be slowly released from the hydrogel for prolonged transfection at the injection site.
In 1990, the direct gene transfer of plasmid DNA into mouse muscles in vivo without the need for a special delivery system was demonstrated^ref. Furthermore, intramuscular inoculation with plasmid DNA encoding reporter genes induced protein expression within the muscle cells. Subsequently, a further study reported the gene expression a year or more after intramuscular injection of plasmid DNA^ref. The basic requirements for the backbone of a plasmid DNA vector are a eukaryotic promoter, a cloning site downstream of the promoter for insertion of transgene(s), a polyadenylation sequence, a selectable marker and a bacterial origin of replication (the Escherichia coli ColE1 ori, which is found in plasmids such as those in the pUC series, is most often used in DNA vaccines because it provides high plasmid copy numbers in bacteria enabling high yields of plasmid DNA on purification)^ref.
A rational approach to improve the efficacy of DNA vaccination or gene therapy would optimise the:
• protein (antigen) expression :
• regulatory elements : In general, virally-derived promoters have provided greater gene expression in vivo than other eukaryotic promoters. In particular, the CMV IE promoter has often been shown to direct the highest level of transgene expression in eukaryotic tissues when compared with other promoters. For example, in one study a plasmid expressing HIV-1 Gag/Env under the regulation of the CMV promoter/enhancer was compared to a comparable plasmid utilising the endogenous AKV murine leukemia LTR^ref. Analysis of the immune responses in macaques injected with the plasmids showed that the CMV-containing plasmid elicited higher Gag- and Env-specific humoral and T-cell proliferative responses, reflecting the greater transcriptional activity of the CMV promoter. Furthermore, it has been demonstrated that inclusion of the CMV intron A improved the level of expression of transgenes expressed by the CMV promoter or other promoter/enhancers^ref. It is thought that the beneficial effect of introns on expression is primarily due to an enhanced rate of polyadenylation and/or nuclear transport associated with RNA splicing^ref. However, some widely used virally-derived promoters, such as the CMV promoter, may not be suitable for some gene therapy applications since treatment with IFN- or TNF-a may inhibit transgene expression from DNA vaccines containing these promoters^{ref1, ref2}. Thus, alternatives to the CMV promoter have been sought. For example, the desmin promoter/enhancer, which controls expression of the muscle-specific cytoskeletal protein desmin, was used effectively to drive expression of the hepatitis B surface antigen priming both humoral and cellular immunity against the antigen^ref. These responses were shown to be of a comparable magnitude to those in mice immunised with comparable DNA vaccines containing the CMV promoter. Other tissue-specific promoters that have been studied include the creatine kinase promoter, also specific to muscle cells^{ref1, ref2}, and the metallothionein and 1,24-(OH)₂-vitamin D₃ dehydroxylase promoters, both of which are specific to keratinocytes^ref. Since the rate of transcriptional initiation is generally increased by the use of strong promoter/enhancers, the rate of transcriptional termination may become rate-limiting^ref. In addition, the efficiency of primary RNA transcript processing and polyadenylation is known to vary between the polyadenylation sequences of different genes. Thus, the polyadenylation sequence used within a DNA vaccine may also have significant effects on transgene expression. For example, it was demonstrated that the commonly used SV40 polyadenylation sequence was less efficient than the minimal rabbit ?-globin and bovine growth hormone polyadenylation sequences in mouse liver, although addition of a second SV40 enhancer downstream of the SV40 polyadenylation signal did increase expression to a level comparable to the other signals^ref. Therefore, it is possible that the strategy of inserting a second SV40 enhancer downstream of a SV40 polyadenylation sequence may be utilised in the construction of more efficient vectors.
• sequences flanking the AUG initiator codon within mRNA influence its recognition by eukaryotic ribosomes. As a result of studying the conditions required for optimal translational efficiency of expressed mammalian genes, the 'Kozak' consensus sequence has been shown to be important^{ref1, ref2}. It has been proposed that this defined translational inititiating sequence (-6 GCCA/GCCAUGG +4) should be included in vertebrate mRNAs located around the initiator codon^ref. It has also been suggested that efficient translation is obtained when the -3 position contains a purine base or, in the absence of a purine base, a guanine is positioned at +4^ref. Prokaryotic genes and some eukaryotic genes do not possess Kozak sequences. Therefore, the expression level of these genes might be increased by the insertion of a Kozak sequence.
• codon usage bias : differences between codon usage in a heterologous gene and the host organism may negatively affect expression from a DNA vaccine vector in vitro and in vivo. Codon optimisation level (codon re-engineering) for mammalian cells have been shown to correlated well with in vitro translational efficiency and in vivo increased antibody titres and CTL reactivity compared to the wild-type sequence of :
• CTL epitopes derived from the intracellular bacterium, Listeria monocytogenes, and the parasite Plasmodium yoelii^ref.
• HIV-1 Gag^ref, gp120^{ref1, ref2}, and gp120 + gp41^ref
• epitope of listeriolysin O^ref1
• receptor-binding domain of the 175-kDa Plasmodium falciparum erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the P. falciparum merozoite surface protein 1 (MSP-1(42))^ref
• fragment C (TetC) of tetanus toxin^ref
• mycobacterial antigen Ag85B : DNA vaccine with hAg85B induced stronger T_h1-like and CTL immune responses in BALB/c mice and generated higher protective immunity in a BALB/c mouse model of Mycobacterium tuberculosis aerosol infection than did the DNA vaccine with wild-type Ag85B. Therefore, codon optimization of mycobacterial antigens (e.g., Ag85B) could improve protein expression and thereby enhance the immunogenicity of DNA vaccines against M. tuberculosis^ref
• the weaker SV40 promoter has been used rather than the CMV IE promoter to drive expression of antigens that induce cell death upon overexpression^ref.
• tissue-specific expression systems may be able to produce stable expression by reducing the probability of inducing an immune response to the transgene. It may be possible to design vectors for gene therapeutic purposes that avoid inducing unwanted immune responses against the encoded antigen by using tissue-specific promoters^ref. Restricting the site of expression of genes should minimise the risks related to aberrant expression of a gene product.
• immune responses to other sequences in the plasmid backbone
• vector backbone DNA sequence modified to enhance immunogenicity via the manipulation of the DNA to include certain immuno-stimulatory sequences (ISS), so that the DNA itself will have an adjuvantising effect. DNA vaccine vectors contain many CpG motifs (consisting of unmethylated CpG dinucleotides flanked by 2 5' purines and 2 3' pyrimidines) that, overall, induce a T_h1-like pattern of cytokine production via binding to TLR9^ref, and are thought to account for strong B-cell activation^ref and CTL responses frequently seen following DNA vaccination^ref. It is possible to augment responses to DNA vaccine vectors by incorporating CpG motifs into the DNA backbone of the plasmid^ref. Transcription of transgenes under control of viral promoters can be down-regulated in the cells subject to the ISS response, probably as a result of the molecular responses to ISS, i.e. the induction of IFN-g. Cytokine induction appears to be mediated by the unmethylated CpG sequences since methylation of plasmid DNA significantly decreases the cytokine levels. Consequentially, the presence of ISS is counterproductive to the expression of proteins. Injection (intravenous) of ODNs formulated in lipid-protamine-DNA complexes (LPD) into mice triggered production of proinflammatory cytokines including interferon gamma and TNF-a. The potency of CpG-containing ODNs in cytokine induction was affected by its flanking sequences and was significantly reduced when CpG was methylated. Preinjection of ODN-containing LPD led to inhibition of transgene expression in lungs after a subsequent injection of LPD containing plasmid expression vector with luciferase gene. The degree of inhibition correlated with the levels of ODN-triggered cytokines. Finally, intraperitoneal injection of dexamethasone suppressed LPD-induced cytokine production, and led to significantly higher levels of transgene expression on both first and second injection. These studies suggest that mutation of potent CpG motifs in plasmid DNA together with the use of immune suppression agent may represent an effective approach to improve cationic lipid-mediated gene transfer to the lung.^ref. Whatever fascinating opportunities ISS may offer, it can be assumed that the exclusion of ISS from expression vectors is beneficial to the objective of delivering a maximum of expression. Another much more worrying topic is the possibility of ISS sequences breaking tolerance to autoantigenes. The strong induction of CTL responses as a result of ISS administration will probably lead to a much-increased awareness of the immunological effects of vectors.
• selection markers : the stimulation of a specific immune response is an attractive goal in cancer therapy. Gene transfer of co-stimulatory molecules and/or cytokine genes into tumor cells and the injection of these genetically modified cells leads to tumor rejection by syngeneic hosts and the induction of tumor immunity. However, the development of host immune response could be either due to the introduced immunomodulatory genes or due to vector components. In this study, human RCC cell lines were modified by a retrovirus to express the co-stimulatory molecule B7-1 together with the hygromycin/thymidine kinase fusion protein (HygTk) as positive and negative selection markers. These B7-1-transduced renal cell carcinoma cell lines were able significantly to activate allogeneic T cell proliferation. The cytolytic activity of these T cells was determined by employing several transduced and nontransduced renal cell carcinoma cell lines as targets. Evidence for a strong vector-specific T cell reactivity induced by the Hyg/Tk protein was obtained in autologous renal cell carcinoma systems. Antibody blocking experiments as well as peptide binding assays demonstrated an HLA-B7-restricted T cell response directed against both the Hyg and the Tk genes. Thus, the vector itself may mask the generation of immune reactivity against tumor antigens and may even detract from it. Vectors with immunogenic potential may be useful for tumor vaccination via cross priming in vivo, whereas antivector reactivities would be detrimental in situations where gene defects are being corrected and where long term expression of a therapeutic protein is required^ref.
• minimalistic, immunogenically defined gene expression (MIDGE^®) vectors (source : Mologen GmbH, Berlin, Germany) for DNA vaccination are linear, covalently-closed vectors containing all the essential information for gene expression (a CMV IE promoter, the gene(s) of interest inserted downstream of the CMV promoter between the restriction sites KpnI and SacI (SsfI), and a SV40 polyadenylation site in pMol, a pUC19 derivative) and none of the non-essential and potentially dangerous plasmid backbone sequences (leaky bacterial promoters and sequences coding for antibiotic selection markers, bacterial origins of replication and other sequence motives which in bacteria tend to mediate integration or recombination). Single-stranded hairpin 5'-phosphorylated oligodesoxy-ribonucleotides (ODN) of sequence GTTCTTCGGG GCGTTCTTTT TTAAGAACGC CCC and GAAGAACGTT TTCCAATGAT TTTTCATTGG AAAAC (self-complementary sequence and separated by 4 thymidine bases (T4)) were purchased from TIBMolBiol (Berlin, Germany) (fig. B) and ligated with T4 DNA ligase (MBI Fermentas, Vilnius, Lithuania). After digestion of remaining product and chromatographic purification, the ODN are concentrated by precipitation in ethanol and sodium-magnesium acetate and resuspended in phosphate-buffered saline. Annealing of the self-complementary ODN sequence results in the formation of a double-stranded stem with an EcoRI overlap on one side and a T4 loop on the other side. The expression cassette is excised from the pG plasmid by an EcoRI digest (250 U/mg, 37°C, overnight). ODNs are added to the resulting solution and incubated with T4-DNA ligase at 25 U/mg and 37°C overnight to stabilize the linear vector against exonuclease activity. Vector backbone remnants are digested by HindIII (500 U/mg) and T7 polymerase (150 U/mg) at 37°C overnight. The final construct is separated from shorter ODN ligation products and nucleosides by anion-exchange HPLC (Merck EMD-DMAE) (fig. A) with sodium phosphate (pH 7.0)-0.1 M NaCl and obtained free of contamination by vector backbone, as verified by PCR^ref.


Transfection efficiency as measured qualitatively and quantitatively with eGFP was found to be comparable to that of the corresponding plasmids. However, hIL-2 secretion and eGFP expression were approximately 2- to 4-fold higher in most cells transfected with these transgenes using MIDGE vectors compared to the plasmid control^ref. MIDGE vectors can also be chemically modified on both ends at defined positions allowing targeting of the DNA to specific cell types or cellular compartments.
• tissue-specific ligands at one end
• a single nuclear localization signal (NLS) at the other end^ref : immunisation of mice with simple and end-modified MIDGE vectors showed that they are efficacious tools to generate and/or manipulate antigen-specific immune responses^ref. MIDGE vectors coding for the LACK antigen confer a highly effective protection against Leishmania infection in susceptible Balb/c mice. Protection is achieved at lower doses of vector compared to conventional plasmids. This efficacy could be greatly improved by the addition of a NLS peptide to the end of the MIDGE vector. In fact, immunization with 2 doses of NLS-modified MIDGE conferred similar or even better protection than that achieved by priming with plasmid DNA followed by boosting with rVV^ref. When a NLS peptide was coupled to the pMOK-HBsAY-MIDGE DNA, HBsAg transfection efficiency in vitro and priming of antibody responses to HBsAg after intramuscular (but not gene gun mediated) injection was enhanced 10- to 15-fold^ref.
Because retroviral vectors are ineffective at transducing nondividing (quiescent) primary cells from human hematopoietic malignancies, nonviral techniques (electroporation and ballistic transfer) using MIDEGE vectors were used to generate B7.1 or GM-CSF-expressing syngeneic Ph⁺ ALL BM185 cell line. Subsequently, protective vaccination experiments with these cells were performed in a leukemia challenge mouse model. Electroporation yielded a high transfection rate (82.6% for B7.1) with moderate GM-CSF secretion/1 x 10⁶ cells (228 pg), whereas ballistic transfer led to a lower transfection rate (30.9%) with high GM-CSF secretion (614 pg). Secondly, mice were immunized with B7.1/IL-2- or B7.1/GM-CSF-expressing BM185 cell vaccines. A better protection of mice that received the B7.1/GM-CSF vaccine was observed compared with these receiving the B7.1/IL-2 vaccine. Protection was additionally enhanced by application of a double stem-loop immunomodulating oligonucleotide containing CpG motifs. Immunization with B7.1/GM-CSF-expressing cell vaccines generated by electroporation and application of double stem-loop immunomodulating oligonucleotide protected mice against a murine Ph⁺ ALL challenge. Ultimately, this approach may also lead to clinical benefit in patients with Ph⁺ ALL^ref. Autologous tumor cells were simultaneously transfected with MIDGE constructs for overexpression of the 2 cytokines IL-7 and GM-CSF and newly designed double stem-loop immunomodulating oligodeoxyribonucleotides (d-SLIM) as a T_h1-promoting and NK cell-stimulating adjuvant. Transfection was performed ex vivo by ballistomagnetic gene transfer. Patients received four subcutaneous injections of at least 1 x 10⁶ of their expression-modulated and immunomodified autologous tumor cells. 10 patients have been enrolled in the study protocol. In all patients no adverse effects could be detected. IL-7 and IFN-g levels were elevated in the serum of the patients after treatment. Interestingly, cytotoxicity of patient-derived PBLs increased significantly during treatment. All 10 patients had progressive disease when entering the protocol. One complete, one partial, and one mixed response with progression of abdominal metastases and regression of lung metastases were observed. 2 patients showed a stable disease after treatment and 5 patients remained in progressive disease^ref. Cats immunized by gen gun with MIDGE vectors encoding either with FIV gp140 and IL-16 or with gp140 and the CpG had greatly reduced viremia^ref
• co-expression of stimulatory molecules or cytokines^{ref1, ref2}
• localisation signals
• intracellular antigen => T_h1 polarization
• secretory signals => secreted antigen => T_h2 polarization^{ref1, ref2, ref3}
• ligand fusions^{ref1, ref2} to direct antigens to sites appropriate for immune modulation
• delivery system used for the vector : a variety of routes of administration of DNA vaccines have been studied, including intramuscular, intradermal, subcutaneous, intravenous, intraperitoneal, oral, vaginal, intranasal and, more recently, non-invasive delivery to the skin^ref
• targeting of the vector for appropriate immune stimulation
The overall level of expression is much lower than with either lipofection or replication-defective viral vectors : naked DNA is also unsuitable for systemic administration due to the presence of serum nucleases. As a result direct injection of plasmid DNA seems destined to be limited to only a few applications involving tissues that are easily accessible to direct injection such as skin and muscle cells. Effectiveness may be improved with physical methods to provide motive force in the delivery of plasmid DNA :
• electroporation (EP) / electropermeabilization / electrotransfer / electric gene transfer : application of a pulsed electric field using a 1-cm² tweezertrode or a stainless stell razor blade electrodes fixed in parallel enhances cytoplasmic membrane permeability (e.g. 50-1500 V / cm, 0.1-75 ms per pulse, 925-ms interval, for 5-10 pulses) and interstitial transport of DNA (e.g. 465 V / cm, 50 ms per pulse, for 10 pulses), whose mobility is inversely correlated with the collagen content of the tissue. Dose of DNA, electrode shape and number, electrical field strength and duration have to be optimized : e.g. use of a syringe electrode allows using much lower electric field strength than that of conventional electrode hence minimizing tissue damage. High ionic strength in the medium is also favorable for gene expression in the skin. Muscle is also a good candidate for electroporation. The mechanism of transport impairment is 2-fold :
• the diffusion coefficient of large molecules is small and the time required for diffusion is porportional to the square of the diffusion distance
• convective transport in solid tumors is limited, due to uniformly elevated interstitial fluid pressure
• in vitro electroporation :
• Axoporator™ 800A, a micropipette-based system in which molecules to be delivered to a cell are loaded into a small electrolyte-containing capillary. When you expose the cells to the electric field, the current is actually an ionic current inside the capillary. Not only does this method allow users to target individual cells, but it also helps them avoid one of the main pitfalls of bulk electroporation: cytotoxicity. The electric shock used to make the membrane porous can also kill the cell^ref. But the Axoporator boasts an unusually high (80%) cell viability rate, largely because the micropipette comes in contact only with a very small portion of the cell membrane. Further, because the system's electrodes are relatively far from the sample, cells are not exposed to harmful chemicals generated by the electric field^ref.
• Excellin's CellArray™ chip is an array-based electroporation device for high-throughput single-cell transfection (up to 500 cells at a time) : it is an electrode-containing silicon flow-through micro-electroporation chip composed of 2 chambers separated by a dielectric membrane. The membrane contains a hole in which a single cell can be seated; electrical current passing through the membrane passes only through the cell, inducing electroporation.  The process is extremely gentle compared to traditional methods, applying only millivolts of electrical field to each cell; it can also be used to probe cell viability. The system continuously detects electrical resistance across cell membranes and can be used to monitor cell health in real time. The CellPort™, a high-volume version of the Excellin system capable of electroporating millions of cells simultaneously, is currently in beta testing.
• Cellectricon is currently developing a capillary electroporation array with the technical features of the Axoporator and the throughput of Excellin's instrument.
Procedure^{ref1, ref2, ref3, ref4} :
• preparation of the cells
• 1| Collect the cells to be transfected from cultures in the mid- to late-logarithmic phase of growth. Use either a rubber policeman or trypsin to release adherent cells. Centrifuge at 500g at 4 °C for 5 min.
• 2|Resuspend the cell pellet in 0.5X volume of the original growth medium and measure the cell number using a hemocytometer.
• 3| Collect the cells by centrifugation, as described in Step 1 and resuspend them in growth medium or phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 2 mM KH₂PO₄) at 15–25 °C at a concentration of 2.5 x 10⁶ to 2.5 x 10⁷ cells/ml.
• 4| Transfer 400-l aliquots of the cell suspension (10⁶–10⁷ cells) into as many labeled electroporation cuvettes as needed. Place the loaded cuvettes on ice.
• 5| Set the parameters on the electroporation device. (A typical capacitance value is 1,050 mF.) Voltages range from 200 to 350 V, depending on the cell line, but generally average 260 V. Use an infinite internal resistance value. Discharge a blank cuvette containing PBS at least twice before electroporating cells.
• introduction of the DNA
• 6| Add 10–30 mg of plasmid DNA in a volume of up to 40 ml to each cuvette containing cells. (Some investigators add carrier DNA (for example, salmon sperm DNA) to bring the total amount of DNA to 120 g.) Gently mix the cells and DNA by pipetting the solution up and down. Proceed to Step 7 without delay. Do not introduce air bubbles into the suspension during the mixing step.
• 7| Immediately transfer the cuvette to the electroporator and discharge the device. After 1–2 min, remove the cuvette, place it on ice and proceed immediately to the next step.
• 8| Transfer the electroporated cells to a 35-mm culture dish using a micropipettor equipped with a sterile tip. Rinse out the cuvette with a fresh aliquot of growth medium and add the washings to the culture dish. Transfer the dish to a humidified incubator at 37 °C with an atmosphere of 5–7% CO2.
• 9| Repeat Steps 6–8 until all of the DNA cell samples have been treated. Recording the actual pulse time for each cuvette will facilitate comparisons between experiments.
• 10| If the objective is stable transformation of the cells, proceed directly to Step 11. For transient expression, examine the cells 24–96 h after electroporation using an appropriate assay.
• 11| To isolate stable transfectants, incubate for 48–72 h in complete medium, trypsinize the cells and replate them in the appropriate selective medium. Change the selective medium every 2–4 d for 2–3 weeks to remove the debris of dead cells and to allow colonies of resistant cells to grow. Thereafter, clone individual colonies and propagate for the appropriate assay.
• in vivo electroporation^{ref1, ref2, ref3, ref4}  was first used after local or systemic DNA injection for skin (transdermal iontophoresis) and liver but skeletal muscle has recently attracted a lot of attention, as expression of the episomal plasmid can be long lived in this tissue. Indeed, a wide range of tissues have been studied including skin, kidney, lung, liver, testes, skeletal and cardiac muscle, joints, spinal cord, brain, retina, cornea and the vasculature.
• effectiveness : in most studies, electroporation increased gene expression by 100- to 1000-fold compared to injection of naked plasmid DNA.
• the exact mechanism by which delivery of plasmid into cells is enhanced is not certain, although it is clear that membranes become effectively permeable once a critical voltage has been achieved (in the order of 200 V/cm in vivo). This was thought to occur by the formation of hydrophilic pores and subsequent movement of plasmid through these pores as a local electrophoretic effect, but when fluorescently labelled plasmid was used to visualize the interaction with single cells in vitro, plasmid was seen to accumulate at the cell membrane via an electrophoretic effect but did not immediately move into the cytosol^ref. Movement into the cytoplasm was relatively slow and continued after the application of the electrical field ended. This is consistent with the DNA active uptake mechanism^ref but may represent a novel physical interaction with the cell membrane as, with time, the plasmid that had not yet entered the cytoplasm became inaccessible to a DNA dye. However, this plasmid accumulation has yet to be observed in vivo and the complex organization of tissues such as muscle may significantly modify this process. A pool of plasmid exists that appears to escape the attention of extracellular nucleases and can be successfully electrotransfered up to 4 h after injection of the DNA^ref.
• electrode design is currently very variable and there is a clear need for good comparative studies.


Meander (a), calliper (b) and needle (c) electrodes used for in vivo electrotransfer. In the case of the meander electrode, an electrical field is generated between each positive and negative electrode with part of the field entering the tissue. This electrode design is the least invasive of the 3. With the calliper electrodes, a near-uniform electrical field is generated between the electrode plates. With needle electrodes, a field is generated between the needles. In some cases, multiple needle electrodes have been used. Skin electroporation using a calliper electrode or a meander electrode is equally effective but the meander electrode appears to be a more patient-friendly design as it avoids the need to pinch the skin between the callipers^ref. For other tissues such as liver and muscle, there is a choice between plate and needle electrodes. In general, the plate electrodes appear to give a more uniform electrical field and are more commonly used for small animals studies but they may not be suitable for electrotransfer in large animals due to the large electrical fields that would need to be applied to larger tissues.
• pattern of electrical pulses also varies considerably between studies ranging from moderate voltage (eg 200 V/cm) pulses of tens of milliseconds to high-voltage microsecond pulses. It is not yet clear what the optimal pattern is but the best results in skeletal muscle were obtained when a single high-voltage pulse was followed by a series of low-voltage pulses^ref. The initial pulse effects an electroporation of the membrane and that the subsequent pulses electrophorese the DNA into the muscle fibre. Currently, the majority of papers appear to use a pattern averaging 6-10 pulses of 20-40 ms at 1 Hz with a field strength in the order of 200 V/cm.
• limitations : there can be substantial damage associated with the procedure and that this can limit the efficiency of transfection^ref. Muscle damage is closely associated with the presence of the plasmid DNA during the electrotransfer and is made worse when the LacZ reporter gene is expressed^ref. Modification of the magnitude and duration of the electrical pulses can ameliorate but not entirely prevent all the plasmid-associated damage following treatment of skeletal muscle.
• applications (electro-gene therapy (EGT)) : when calliper electrodes are applied either side of the thorax of mice after intratracheal instillation of a solution of plasmid DNA, there is substantial short-term reporter gene expression in the lung following this procedure with low mortality in the treated animals. Gene expression was seen predominantly in the peripheral alveoli and in both the epithelium and deeper cells. Histological analysis of the lungs 24 h after treatment did not reveal any obvious signs of damage^ref. Thus electroporation appears a relatively safe procedure in the lungs. Application to humans might be possible using bronchoscope-directed electrodes but would only result in treatment of local areas of the lung. Other recently developed targets for electroporation include joints^ref, spinal cord^ref and the retina^ref. Very high gene transfer efficiencies into retinal cells, in particular the photoreceptors, can be achieved with electroporation in neonatal rats and mice^ref. Electroporation can also be used to deliver oligonucleotides to muscle, specifically for antisense-mediated exon skipping in dystrophic skeletal muscle^ref. Electroporation has been extensively tested for gene transfer into tumours. Even the transfer of empty plasmid vector is sufficient to cause significant tumour regression in some models^ref. Electroporation of plasmids containing IL-12 and IL-18 has been shown to produce a synergistic effect in inhibiting tumour growth, both for the treated tumour and also for the untreated contralateral tumour^ref. In another study, electrotransfer of a combination of bleomycin and a plasmid encoding IL-12 not only produced complete remission in a proportion of mice carrying a subcutaneous melanoma but also provided a significant protective effect against established metastases^ref. Given the success of electrotransfer of bleomycin in many clinical trials^ref, the combination with gene transfer appears to offer further potential in the delivery of a clinically effective treatment. A combination of vascular delivery with electroporation for the liver has been used^ref. Intravenous administration of plasmid via the tail vein in the mouse enhanced gene expression in the liver following electroporation compared to direct injection into the liver before electroporation. Expression was further improved by temporarily blocking blood flow through the vena cava or the portal vein and hepatic vein. It remains to be seen if this combination approach will work in other tissues. Electrotransfer can also be used for genetic vaccination, and a number of laboratories have demonstrated substantial improvements in responses to a variety of antigens^{ref1, ref2, ref3}. Electrotransfer of plasmid DNA encoding immunoglobulins might be an alternative to the administration of monoclonal antibodies in the treatment of a variety of conditions and have demonstrated prolonged expression in both mice and sheep^ref. An interesting development of in vivo electroporation for research purposes has been the application of this technology to the developing central nervous system (CNS) of rodent embryos^{ref1, ref2, ref3}. Using very fine electrodes, it is possible to deliver plasmids to defined regions of the developing brain to track cell migration and to modify patterns of development. Recently, this technology has been extended to adult rodents, showing that it is possible to express selectively plasmid-based constructs in specific regions of the adult CNS^ref.
• integration of plasmid DNA has not been previously reported following direct intramuscular injection despite a number of careful studies from several laboratories^ref. In contrast, integration of plasmid DNA can be detected following electroporation of skeletal muscle in vivo. However, this is still a very rare event, below the level of background genomic mutation, and so is unlikely to be a significant safety risk with respect to clinical application.
Nonviral gene delivery mediated by electroporation has been shown to be efficient in different tissues including skin. There are no detailed reports about the effects of electroporation on skin tissue, when used for gene/DNA vaccine delivery. The efficacy of skin targeted DNA vaccine delivery using electroporation in rabbits has been demonstrated^ref, and the safety aspects of the electroporation technique in vivo in rabbits have been investigated. Different electroporation parameters (100-300V) were tested for their effects on skin viability, macroscopic barrier property, irritation and microscopic changes in the skin. Skin viability was not affected by the electroporation protocols tested. The electroporation pulses induced skin barrier perturbation and irritation as indicated by elevated transepidermal water loss (TEWL) and erythema/edema, respectively. Microscopic studies revealed inflammatory responses in the epidermis following electroporation using 200 and 300V pulses. However, these changes due to electroporation were reversible within a week. The results suggest that the electroporation does not induce any irreversible changes in the skin and can be a useful technique for skin targeted DNA vaccine delivery^ref
• magnetofection : application of a magnetic field to cells transfected with plasmid DNA mixed with superparamagnetic nanoparticles following local injection has been reported to enhance in vivo gene transfer to the gastrointestinal tract and the vasculature of the ear^ref. The improved gene transfer associated with magnetofection does not appear to be as substantial as that seen with hydrodynamic delivery or electroporation, although direct comparisons have not yet been made.
• hydrodynamic injection, a rapid injection of a large volume or naked DNA solution (eg 5 mg plasmid DNA injected in 5-8 s in 1.6 mL saline solution for a 20 g mouse) via the tail vein, can induce potent gene transfer in internal organs, expecially the liver because of its flexible structure which can accommodate large volume of solution. The hydrostatic pressure breaks the endothelial barrier and forces DNA into the liver cells before it is mixed with blood. Pressurized vascular delivery improves plasmid transfection in vivo with a more widespread distribution than local injection. As plasmid DNA contains no proteins for attachment to cellular receptors, naked plasmid has no specific targeting to different cell types and thus it is essential that the DNA is placed in close contact with the desired cell type. The use of pressure to achieve this close contact has also dramatically increased the efficiency of delivery, in particular to liver and skeletal muscle. The use of rapidly delivered extremely high volume (equivalent to total blood volume) injections via the tail vein of the mouse leads to highly efficient transfection of hepatocytes throughout the majority of the liver. A series of very recent papers have attempted to explain the physical basis for this phenomenon. Hydrodynamic gene delivery leads to a transient decrease in heart function and a rapid rise in venous pressure that leads to enlargement of the fenestrae in the liver sinusoids^ref. This in turn allows hydrostatic pressure to act on the hepatocyte cell membranes causing transient pores or defects, a process they term hydroporation. The dynamics of the transfection process support this concept of transient hyperpermeability of the hepatocyte cell membrane^{ref1, ref2, ref3} and do not support the active uptake mechanism^ref. The use of this hydrodynamic technique in the mouse has allowed comparison of the effect of different vector sequences^ref, the testing of optimal expression constructs^ref and the efficient delivery of siRNA to the liver^{ref1, ref2}. The same technique has been applied to rats^ref. However, this approach, while useful for studies in rodents, is not directly applicable to humans. Local hydrodynamic delivery into specific lobes of the rabbit liver using balloon cuff catheters can be safely achieved with this approach without the need for surgery to expose the liver^ref. As with the tail-vein injections, there were only transient elevations in liver-specific enzymes released into the blood. Pressure-mediated plasmid delivery has also been used for other tissues, such as the kidney^ref. The importance of local hydrostatic pressure has been shown : mechanical massage of the liver increases gene transfer following intravenous delivery of plasmid DNA^ref. Plasmid has been successfully delivered to the diaphragm using the venous approach with temporary occlusion of the cranial vena cava^ref. For delivery to limb musculature, pressure-mediated plasmid delivery is generally performed via the arterial system. This approach has been used for delivery of plasmid to multiple muscles in the temporarily isolated limbs of primates^ref, and isolated limb perfusion is an established technique in clinical practice for the treatment of tumours^ref. Plasmid uptake and expression was more efficient than for direct intramuscular injection and was detected in all muscles of the perfused limb. There have been no subsequent publications using this gene delivery methodology, although unpublished studies were reported^ref. Expression of human minidystrophin has been observed (using a species-specific antibody) in up to 30% of muscle fibres in the limb muscles of rats following pressure-mediated arterial delivery (Fletcher, Wells and Wells, unpublished results). The same technique is currently being used to deliver dystrophin to dystrophic muscle in the legs of the canine model of X-linked muscular dystrophy. It may not be necessary to use the arterial route to deliver plasmid to skeletal muscle^ref : there is remarkably high efficiency delivery of a dystrophin plasmid to dystrophic mouse muscle by both arterial and venous routes provided that there was a temporary occlusion of the blood supply that increased pressure within the muscle vascular bed.
• external pressure (e.g. external mechanical massage of the liver increases gene transfer following intravenous delivery of plasmid DNA^ref) : mechanical stretching of the endothelial barrier may affect uptake of DNA into cells.
• gene gun (GG) / biolistics / bioballistic / particle bombardment-mediated gene transfer / accelerated particles gene delivery or particle bombardment : coating of DNA to be delivered into cells onto extremely small carrier particles, which are designed to be small in relation to the cells sought to be transformed by the process. The DNA sequence containing the desired gene can be simply dried onto a small inert particle. The particle / microprojectile may be made of any inert material such as
• inert metal (gold, silver, platinum, tungsten, etc.)
• inert plastic (polystyrene, polypropylene, polycarbonate, etc.).
Preferably, the particle is made of gold, platinum or tungsten. Most preferably, the particle is made of gold. Suitably, the particle is spherical and has a diameter of 0.5 to 5 mm, preferably 1 to 3 mm. DNA molecules in such a form may have a relatively short period of stability and may tend to degrade rather rapidly due to chemical reactions with the metallic or oxide substrate of the particle itself.
Thus, if the carrier particles are first coated with an encapsulating agent, the DNA strands have greatly improved stability and do not degrade significantly even over a time period of several weeks :
• a suitable encapsulating agent is polylysine (molecular weight 200,000) which can be applied to the carrier particles before the DNA molecules are applied. Other encapsulating agents, polymeric or otherwise, may also be useful as similar encapsulating agents, including spermidine. The polylysine is applied to the particles by rinsing the gold particles in a solution of 0.02% polylysine and then air drying or heat drying the particles thus coated.
Once the metallic particles coated with polylysine were properly dried, DNA strands are then loaded onto the particles. The DNA is loaded onto the particles at a rate of between 0.5 and 30 mg of DNA per mg of gold bead spheres. A preferable ratio of DNA to gold is 0.5-5.0 mg of DNA per mg of gold. DNA, spermidine-free base and CaCl₂ are sequentially added. For a 10 µl shooting (0.5 mg of particles) 4 µl of DNA solution (generally 1 µg/ µl), 4 µl of 0.1 M or 1 M spermidine-free base and 10 µl 2.5 M CaCl₂ are added. In a typical vaccination, each cartridge contains a 0.5 mg gold coated with 0.6-3 mg of plasmid DNA.
A sample procedure begins by loading plasmid DNA coated onto particles into a g irradiated (preferably about 30 kGy) tefzel tubing, which is subsequently cut into 1.27 cm lengths to serve as cartridges and stored desiccated at 4°C until use. After resuspension, the desired aliquot of particles is removed and washed 3 times with distilled water in a Treff Lab microtube. A 400-500 psi He pulse is used to accelerate the colloidal solution into a wide range of biological samples from cellular organelles to in situ mammalian organs (most commonly shaved abdominal skin)^ref. There are various systems most of which use a burst of a gas, such as dry steam generated by an electric discharge on a drop of water, a capacitive electric discharge through a wire electrode that instantaneously vaporizes creating a shock wave, a gun powder explosion, a burst of compressed helium or a gas flow^ref. In the case of a gas flow, particles are directly propelled whereas in the case of a gas burst the kinetic energy is transmitted to the particles that are loaded on an accelerated macrocarrier. The sudden stopping of the macrocarrier projects the micro particles by the inertia principle. The highest levels of both protein expression and antibody production are correlated with particle delivery to the epidermis while deliveries extending into the dermis result in decreased protein and antibody production. Optimal immune responses are also shown to be dependent upon the delivery of a sufficient number of DNA-coated gold particles, indicating that a dose-response relationship exists between the number of particles delivered and the resultant protein expression and antibody production^ref. Applications have focused on into silkmoth^ref, Xenopus^ref, Drosophila^ref, Artemia fransciscana and Anopheles gambiae embryos, neurones (that have proved difficult to transfect by conventional means).
Diolistic transfection : delivery of both DNA and lipophilic dyes^ref
• PowderJect-XR™ (formerly Accell^®, PowderJect Vaccines Inc., WI, USA)
• PDS/1000-He^® (BioRad, Hercules, CA, USA) system is a helium burst that uses a macrocarrier system device. The macrocarrier consists of a floppy, thin kapton disk (45 µm in thickness) that is accelerated by a compressed helium burst depending on the value of a rupture disk. The calibrated thickness of the rupture disk determines the value of the shooting pressure, which in turn determines the velocity and the penetration of the microprojectiles.



However, the higher the shooting pressure, the higher the expanded helium volume correlated with the deleterious blast effect. In this system, high shooting pressure could contradict the expected penetration effect. The blast effect lies in the structure of the macrocarrier of the BioRad device, which crashes into the stopping screen at the end of its path. At this moment, a strong residual helium flow is generated, which destroys, or blows out, fragile target samples. Moreover, the particle impact is systematically decentered due to poor mechanical guidance of the fine macrocarrier, the flight of which is always off course. Mechanical modifications on the Bio Rad PDS/1000 He biolistic device shooting module. A: Schematic illustration of the problems encountered with the original shooting module. B: Decentering of the shootings with the original shooting module. C: Schematic illustration of our mechanical modifications. D: Focusing the shootings with our shooting module. E: Plan of our macrocarrier mould (left) and the shooting module (right).

Advantages :
• practicality of targeting ... :
• skin : ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell death is dependant on the number of micro-particles delivered per unit of tissue surface area. Micro-particles densities of 0.16+/-0.27 (mean+/-S.D.), 1.35+/-0.285 and 2.72+/-0.47 per 1000 mm² resulted in deaths of 3.96%+/-5.22, 45.91%+/-10.89, 90.52%+/-12.28, respectively. These results suggest that optimization of transfection by genes administered with gene guns is - among other effects - a compromise of micro-particle payload and cell death^ref
• mucosae, to generate protective immunity against mucosal pathogens : intraoral administration of DNA in the cheek, using a jet immunization technique, elicited the highest IgA mucosal responses. Intranasal immunization gave strong mucosal IgA responses and persistent systemic IgG. Immunoglobulin isotype analysis revealed an IgG₁ profile for intramuscular tongue and gene gun immunizations and an IgG_2a profile following oral jet injection and intranasal application^ref
• oral mucosa^{ref1, ref2}
• ventral surface of the tongue^{ref1, ref2}
• anorectal epithelium^ref
• genital mucosa^ref
• female genital tract^{ref1, ref2, ref3} for contraception and the prevention of sexually transmitted diseases
• direct delivery through the cell membrane into the cytoplasm and even the nucleus, bypassing the endosomal compartment^ref
• maximal protein expression and associated antibody titers are elicited with the use of 3-4 orders of magnitude less DNA than is typically required in direct DNA inoculation studies (1 mg by gene gun is equivalent to ~ 100 mg injected)^{ref1, ref2}
Disadvantages :
• deleterious blast effect on fragile insect tissues, such as embryos, oocytes and imaginal wing disks (reduced when the original floppy macrocarrier is replaced by a rigid macrocarrier to avoid the effects of the helium blast^ref)
• shallow penetration of DNA into the tissue (the efficiency of the gene gun bombardment is reinforced by the addition of a focusing nozzle^ref)
This approach has been reported to be superior to other methods of plasmid delivery to the skin^{ref1, ref2} but tends to generate a T_h2 (humoral) rather than the T_h1 (cytotoxic) immune response that commonly follows intramuscular administration^{ref1, ref2}. This difference in the nature of the immune response can be modified by different boost strategies following the initial priming vaccination by gene gun delivery of plasmid DNA^ref. Some reports have claimed that the route of DNA delivery has a major impact on the type of immune responses induced by DNA vaccines in mice, with GG inducing T_h2 biased immune responses dominated by IgG₁ subclass antibodies and IM inducing T_h1 biased responses dominated by IgG_2a antibodies^{ref1, ref2, ref3}. However, most studies of the effects of route of administration for DNA vaccination have been performed with viral antigens : this pattern was observed for only 1 of 4 Brugia malayi antigens tested (BM5). Other recombinant plasmids produced antibody responses biased toward IgG₁ or IgG_2a independent of route. These results suggest that the isotype profile of antibodies generated by DNA immunization depends not only on the route of DNA delivery but also on the intrinsic nature of antigens used for immunization^ref. Apart from antibody levels, several studies have reported that the route of DNA delivery can affect the protective efficacy of DNA vaccines, which does not always correlate with antibody titers. For example, DNA encoding a Plasmodium yoelii antigen (HEP17) protected mice equally well when given by the GG or IM routes, while DNA encoding two other antigens (CSP and SSP2) induced better protection by the IM route than by the GG route^ref. On the other hand, DNA encoding CSP was protective in a different malaria model only after GG vaccination^ref. Thus, neither route was conclusively favored in all cases; different injection methods may provide optimal immunogenicity for different antigens. Therefore, our results and the work of others suggest that it is not possible to reliably predict the optimal route for a given antigen to achieve desired immune responses. There is currently no substitute for empirical testing of each antigen. Co-injection of multiple plasmids successfully induces antibody to each component antigen encoded by the plasmid mixture^ref. Bombardment of tissues by plasmid-coated microparticles has previously received considerable attention for gene transfer in animals after originally being developed for gene delivery to plants. The application of the gene gun technology in animals has been limited to superficial tissues such as the skin. Recent papers have continued to use this approach for applications such as gene therapy of bladder pain using POMC gene transfer in a rat model^ref, the transfer of IL-10, IL-12 and B7.1 into murine tumors^ref, gene transfer into the heart^ref, gene transfer into mouse embryos^ref and treatment via the skin of a mouse model of glycogen storage disease type II^ref. A new design of gene gun delivers microparticles at a higher pressure, thus accessing subcutaneous tissues, such as muscle or tumours, and consequently achieving longer-term gene expression^ref.
Data presented to date has been generated using a research device known either as the PowderJect XR system or the Genegun. Using these systems DNA loaded gold particles are coated onto the inside of short tubes and an external gas source is required to deliver the drug product to the epidermis. Although efficient for experimental purposes, this system is not suitable for commercial use. A commercial device system was thus required and PowderMed have named this the PowderJect ND system. In the ND system, DNA loaded gold particles are filled into a cassette that acts as a closed primary drug product container.  No changes are required to the DNA on gold formulation process. The cassette is inserted into the device during the final product assembly and packaging process, thus producing a convenient single dose disposable commercial product. On actuation, the release of helium from a self-contained micro-cylinder propels the particles from their stationary state in the cassette through the nozzle and towards the skin surface at high pressure and at near supersonic speeds. The system is vented via a silencer such that the resulting backpressure at the skin surface is less than 5 bar. The ND system has now been clinically tested demonstrating that the immunogenicity and reactogenicity of the commercial ND system is non-inferior to the XR system. All future PowderMed clinical projects will thus use the commercial ND system. The PowderMed technology is currently in phase I trials using three different drug products: HSV, non-small cell lung cancer and HIV.
A unique biomedical device, the venturi powdered injection system (venturi), is proposed for the epidermal delivery of DNA vaccines. The novelty of this hand-held venturi device is in using the venturi effect to entrain DNA-coated micro-particles into an established quasi-steady transonic helium flow and accelerate them to an appropriate momentum for penetrating the skin or mucosal tissue to achieve an immunological effect. Computational fluid dynamics (CFD) has been employed to scrutinize an experimental venturi device. Key features of gas dynamics and gas-particle interactions have been presented. A parallel extension was added to improve the uniformity of gas and particle flow for a better particle penetration distribution. The overall capability of the venturi biolistic configuration for delivering micro-particles has been explored and discussed. Statistical analysis has shown that the modelled micro-particles have achieved a mean velocity of 654m/s for intracellular DNA vaccine delivery applications^ref.
• osmotic shock
• femtosecond focused laser pulses / laser beam gene transduction (LBGT) / laser induction^ref. Gene delivery into muscle could be enhanced by application of a femtosecond infrared laser (5 s at 30 mW) and they compared this to in vivo electroporation of the same quantity of DNA into the same muscle (16 x 20 ms pulses at 200 V/cm). The authors concluded that there was a similar efficiency of gene delivery and that this was accompanied by substantially less damage when using LBGT. The mechanism by which the laser enhanced gene transfer was not clear but likely involved local disruption of the muscle cell membrane. 2 issues arise from this work. The comparison with electroporation was not unexpected as several other groups have shown that multiple pulses at 200 V/cm cause substantial muscle damage and that modifications to the protocol can reduce this damage^{ref1, ref2}. Secondly, the laser was focused to 2 mm under the skin whereas substantially deeper focusing would be required for percutaneous delivery to human skeletal muscle. It remains to be seen if LBGT can be significantly scaled up in studies of larger muscles or other tissues.
• ultrasounds (US) (sonophoresis) can increase the permeability of cell membrane to macromolecules such as plasmid DNA. Combination of the microbubbles with US could further increase the gene expression level. Ultrasound contrast agents (UCAs), lower the energy for cavitation by US energy. In most cases, perfluoropropane-filled albumin microbubbles (Optison^®) are used as microbubbles. It is modified with plasmid DNA before injection. Clinically applicable ultrasound can enhance plasmid-based gene delivery in vivo. In vivo electroporation- and pressure-mediated delivery, although very efficient, are rather invasive techniques. Consequently, other methods have been examined for their ability to provide motive force in the delivery of plasmid DNA. Ultrasound is in general clinical use for both therapeutic and diagnostic purposes. Low-level ultrasound is used for diagnostic imaging, ultrasonic shock waves are used in the treatment of kidney stones (lithotripter) and high-intensity focused ultrasound (HIFU) is used for the thermal destruction of tumours. A number of groups have demonstrated the utility of these various modalities of ultrasound in enhancing the delivery of plasmid DNA. For example, Taniyama et al have used low-dose (1 min at 1 MHz, 2.5 W/cm²) ultrasound to improve gene delivery in skeletal muscle^ref and the carotid artery^ref. Likewise, other groups have used a similar approach for gene transfer into skeletal muscle^{ref1, ref2, ref3} and the heart^{ref1, ref2}. In contrast, Huber et al^ref used HIFU (1 min at 0.85 MHz, 155 W over a 2 mm width) to perform gene transfer to the carotid artery on the grounds that this modality allows specific localization of the enhanced gene transfer. They demonstrated an eight-fold increase in total reporter gene expression with HIFU alone and a 17.5-fold increase when ultrasound contrast bubbles were used. However, the reported increase in transfection with contrast enhanced HIFU is not significantly different from the fold increases shown in many of the lower power delivery ultrasound studies. HIFU and lithotripter shock waves have been used to simultaneously ablate tumours and perform gene transfer into the residual mass but gene expression was very variable, possibly due to variable degrees of tumour ablation^ref. The application of ultrasound results in acoustic cavitation that can disrupt tissues and produce transient membrane permeabilization, thus enhancing the delivery of plasmid to the cytoplasm. Nucleation agents such as ultrasound contrast agents can enhance cavitation, and several studies have examined a range of such contrast agents, concluding that albumin-coated octa-fluoropropane gas microbubbles (Optison) is preferable to several other commonly available agents^{ref1, ref2}. Electron micrographs of cells treated with Optison and ultrasound in culture that suggest that transient pores in the cell membrane are indeed opened up immediately following treatment^ref. Even with the use of ultrasound contrast reagents, most studies only show an average of 10- to 15-fold increase in reporter gene expression, which is considerably below that which can be achieved with electroporation. A combination of electroporation and ultrasound (electro-sonoporation) was superior to either electroporation or ultrasound alone^ref. However, it is difficult to compare this study to others in the field due to insufficient methodological details for the electroporation component.
Delivery to the skin :
• gene gun (particle mediated epidermal delivery (PMED))
• electroporation
• laser induction
• sonophoresis
• microporation : a vaporization process is used to remove tiny areas of the stratum corneum creating microscopic pores that allow access to the underlying viable epidermis. Reporter gene expression was 100-fold increased following application of an adenovirus vector to microporated skin when compared to intact skin. Furthermore, 10-100-fold greater cellular and humoral immune responses were observed following topical administration of an adenovirus vaccine to microporated skin versus intact skin. Hairless mice responded to the microporated adenovirus vaccine equivalently to mice with normal hair follicle distribution demonstrating the activity of the microporated vaccine was not related to follicle count. In a tumor challenge model using a surrogate antigen, microporation increased vaccine efficacy by approximately 100-fold compared to intact skin. Finally, microporation enabled delivery of an adenovirus vaccine carrying a relevant melanoma antigen resulting in the development of auto-immune vitiligo and tumor protection^ref
• microcannula that restricts delivery to the dermis and solid arrays of structurally precise, micron-scale silicon projections (microenhancer arrays (MEAs)) specifically designed to mechanically disrupt the stratum corneum and provide direct access to the epidermis with minimal associated discomfort and skin irritation. In a mouse model, MEA-based delivery enabled topical gene transfer resulting in reporter gene activity up to 2,800-fold above topical controls. MEA-based delivery enabled topical immunization with naked plasmid DNA, inducing stronger and less variable immune responses than via needle-based injections, and reduced the number of immunizations required for full seroconversion. Together, the results provide the first in vivo use of microfabricated devices to breach the skin barrier and deliver vaccines topically, suggesting significant clinical and practical advantages over existing technologies^ref.Microneedle-based cutaneous and nasal mucosal delivery of recombinant Bacillus anthracis protective antigen (rPA) has been investigated in mice and rabbits. In mice, intradermal (id) delivery achieved up to 90% seroconversion after a single dose, compared with 20% after intramuscular (im) injection. Intranasal (inl) delivery of a liquid formulation required 3 doses to achieve responses that were comparable with those achieved via the id or im routes. In rabbits, id delivery provided complete protection against aerosol challenge with anthrax spores; in addition, novel powder formulations administered inl provided complete protection, whereas a liquid formulation provided only partial protection. These results demonstrate, for the first time, that cutaneous or nasal mucosal administration of rPA provides complete protection against inhalational anthrax in rabbits^ref
• naked DNA with or without tape stripping and DNA/lipid based complex such as liposomes, niosomes, Transfersomes^®, or microemulsion^ref
Although there are clinical precedents for the use of electroporation, isolated limb/organ perfusion and ultrasound, it still remains to be seen if any of these approaches will be translated into gene therapy clinical trials. The most likely areas of application will be for local administration to tumours and for enhanced genetic vaccination. Previous genetic vaccination studies using naked plasmid DNA have been disappointing^ref. However, electrotransfer into mouse muscle can produce a more dramatic induction of CD8 T cells than any other form of vaccination including the use of live vaccinia virus^ref. Importantly, the use of electroporation maintains a high efficiency of vaccination when pre-clinical studies are moved from small to large animal models^ref. The recent Phase 1 clinical trial studying the safety of plasmid-based gene transfer of dystrophin in Duchenne and Becker muscular dystrophy patients in France has been completed and initial reports confirm that no safety problems were encountered, although as expected the efficiency of gene delivery using direct intramuscular injection was very low compared to similar studies in dystrophic mice. Studies of arterial pressure-mediated gene transfer in dystrophic dogs show promising results and it is hoped that the same technique but involving an individual muscle will be the subject of the next clinical trial. A similar method has also been used to deliver adeno-associated viral vectors to multiple muscles in the haemophiliac dog^ref and the use of physical delivery methods may also prove useful for viral as well as nonviral vectors. Physical enhancement of viral gene delivery has been less widely investigated compared to the delivery of nonviral vectors. Optimal coronary artery delivery of adenoviral vectors requires pressures greater than normal systolic but that the threshold for this effect is limited and trebling the pressure induces marked cardiac damage^ref. Similarly, high pressure was used to deliver an adenoviral vector via a double balloon catheter in the rat coronary artery^ref. Ultrasound has also been used to deliver adenoviral vectors to the heart and was dependent on the presence of liposome microbubbles^ref. Indeed, ultrasound may be used more in the future for viral vectors where it is desirable to target specific tissues
Plasmid-derived DNAs isolated from Escherichia coli are circular duplexes, and this may be one of the reasons for their low expression efficiency, because the proteins involved in transcription may be loaded on to circular DNA less efficiently than on to linear DNA. In addition, the plasmid-derived DNAs contain regions, such as the origin of replication and the antibiotic resistance gene, which are unnecessary for gene expression in mammalian cells and could be deleted. The expression of transgenes on short DNA molecules lacking the extra portions may be efficient. Small, linearized DNA duplexes that are end-capped with a highly stable loop by enzymatic ligation (e.g. 5'-GCGAAGC-3' : dumbbell-shaped DNA) to make the terminus rigid and to prevent digestion by exonucleases have similar endocytosis and nuclear entry but are 5-10-fold more expressed than circular DNAs : smaller linearized DNA dumbbells are more effective than longer ones.
For Bacteria some transformation-competent Escherichia coli preparations exist :
• CaCl₂ transformation
• Ca₃(PO₄)₂ coprecipitation + thermal shock
• Electrotransformation
• Inoue "ultra-competent" method
• JS5 transformation
• RbCl method
Calcium phosphate-mediated transfection of eukaryotic cells^ref : the uptake of DNA is markedly enhanced when the nucleic acid is presented as a coprecipitate of calcium phosphate and DNA^ref. The insoluble precipitate attaches to the cell surface and is brought into the cells by endocytosis. Since the publication of the original method^ref, increases in efficiency have been achieved by including additional steps such as glycerol shock (Parker, B.A. & Stark, G.R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J. Virol. 31, 360–369 (1979)) and/or chloroquine treatment^ref. This protocol is a modified version of a published method4, in which calcium phosphate-based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells were rigorously optimized. The protocol is easily adapted for use with other types of cells, both adherent and nonadherent. Procedure
• transfection of the cells :
• 1| harvest exponentially growing cells 24 h before transfection by trypsinization and replate them at a density of 1 x 10⁵ to 4 x 10⁵ cells/cm² in 60-mm tissue culture dishes or in 12-well plates in the appropriate complete medium. Incubate the cultures at 37 °C for 20?24 h in a humidified incubator with an atmosphere of 5-7% CO₂. Change the medium 1 h before transfection.
• 2| to prepare the calcium phosphate-DNA coprecipitate, combine 100 l of 2.5 M CaCl₂ with 25 g of plasmid DNA in a sterile 5-ml plastic tube and, if necessary, bring the final volume to 1 ml with 0.1X TE (pH 7.6). Mix one volume of this 2X calcium phosphate-DNA solution with an equal volume of 2X HEPES-buffered saline (HBS) at 15-25 °C. Tap the side of the tube to mix and incubate the solution at 15-25 °C for 1 min.
• 3| immediately transfer the calcium phosphate-DNA suspension into the medium above the cell monolayer. Use 0.1 ml of suspension for each 1 ml of medium in a well or 60-mm dish. Rock the plate or dish gently to mix the medium, which will become yellow-orange and turbid. If the cells will be treated with glycerol and/or chloroquine, proceed directly to Step 5.
• 4| incubate the cells at 37 °C for 2-6 h in a humidified incubator with an atmosphere of 5-7% CO₂; then remove the medium and DNA precipitate by aspiration. Add 5 ml of prewarmed (37 °C) complete growth medium to each dish of cells and return the cells to the incubator for 1-6 d. Proceed to Step 9 to assay for transient expression of the transfected DNA or to Step 10 if the objective is stable transformation of the cells.
• treatment of cells with chloroquine :
• 5| dilute 100 mM chloroquine diphosphate 1:1,000 directly into the medium either before or after the addition of the calcium phosphate-DNA coprecipitate to the cells (Step 3). Incubate the cells at 37 °C for 3-5 h in a humidified incubator with an atmosphere of 5-7% CO₂. The concentration of chloroquine added to the growth medium and the duration of treatment are limited by the sensitivity of the cells to the toxic effect of the drug and should be determined empirically for each cell type.
• 6| remove the medium, wash the cells with phosphate-buffered saline (PBS) and add 5 ml of prewarmed complete growth medium to each dish of cells. To further enhance uptake by treatment with glycerol, proceed to Step 7. Otherwise, return the cells to the incubator for 1?6 d. Proceed to Step 9 to assay for transient expression of transfected DNA or to Step 10 if the objective is stable transformation of cells.
• treatment of cells with glycerol
• 7| after cells (chloroquine-treated or untreated) have been exposed to the calcium phosphate-DNA coprecipitate in growth medium for 2?6 h, remove the medium by aspiration and wash the monolayer once with PBS. Add 1.5 ml of 15% glycerol in 1X HEPES-buffered saline to each monolayer and incubate the cells at 37 °C for the predetermined optimum length of time. Because cells vary widely in sensitivity to the toxic effects of glycerol, each cell type must be tested in advance to determine the optimum time of treatment (30 s to 3 min).
• 8| remove the glycerol by aspiration and wash the monolayers once with PBS. Add 5 ml of warmed complete growth medium to each dish of cells and incubate the cells for 1-6 d. Proceed to Step 9 to assay for transient expression of the transfected DNA or to Step 10 if the objective is stable transformation of the cells.
• detection of DNA expression in transfected cells
• 9| to assay for transient expression of the introduced DNA, harvest the cells 1-6 d after transfection. Analyze RNA or DNA using hybridization; analyze the newly synthesized protein by radioimmunoassay, by immunoblotting, by immunoprecipitation after in vivo metabolic labeling or by assays of enzymatic activity in cell extracts
• 10| to isolate stable transfectants, incubate the cells at 37 °C for 24-48 h in nonselective medium to allow time for expression of the transferred gene(s). Trypsinize and replate the cells in the appropriate selective medium, or add selective medium directly to the cells without further manipulation.
• 11| change the selective medium with care every 2-4 d for 2-3 weeks to remove the debris of dead cells and to allow colonies of resistant cells to grow. Then clone individual colonies and propagate for the appropriate assay.
• 12| obtain a permanent record of the numbers of colonies by fixing the remaining cells with ice-cold methanol for 15 min, then staining with 10% Giemsa solution for 15 min at 15-25 °C and rinsing in tap water.
• bacteriophages (or phages) are viruses of bacteria, consisting of nucleic acid packaged within a protein coat. In eukaryotic hosts, phages are unable to replicate and in the absence of a suitable prokaryotic host, behave as inert particulate antigens. In recent years, work has shown that whole phage particles can be used to deliver vaccines in the form of immunogenic peptides attached to modified phage coat proteins or as delivery vehicles for DNA vaccines, by incorporating a eukaryotic promoter-driven vaccine gene within their genome. While both approaches are promising by themselves, in future there is also the exciting possibility of creating a hybrid phage combining both components to create phage that are cheap, easy and rapid to produce and that deliver both protein and DNA vaccines via the oral route in the same construct^ref. The stability of whole bacteriophage l particles, used as a DNA vaccine delivery system has been examined. Phage were found to be highly stable under normal storage conditions. In liquid suspension, no decrease in titre was observed over a 6-month period at 4 and -70°C, and phage stability was unaffected by freeze/thawing. The measured half life of phage in suspension was 36 days at 20°C, 3.4 days at 37°C and 2.3 days at 42°C. Freeze drying of a phage suspension (with or without the stabilizers dry skim milk or trehalose) resulted in 5-20% residual viability. Following desiccation (with or without stabilizers), measured half lives ranged from 20 to 100 days at 20°C, 2.6 to 38 days at 37°C, 2.1 to 26 days at 42°C, 7 to 33 h at 70°C, and 1.3 to 6m at 100°C. In all cases the addition of trehalose significantly increased the stability of the desiccated phage. When stored at -70°C, desiccated phage appeared to be stable in the absence of stabilizers. When phage l was diluted into water, a marginal loss in titre was observed over a 2-week period. Over a 24 h period, liquid phage suspensions were stable within the pH range pH 3-11, therefore oral administration of bacteriophage DNA vaccines via drinking water may be possible^ref. Mice and rabbits have been vaccinated with whole bacteriophage l particles containing a DNA vaccine expression cassette under the control of the CMV promoter (enhanced green fluorescent protein [l-EGFP] or hepatitis B surface antigen [l-HBsAg]). Mice were vaccinated twice intramuscularly (i.m.) with 5x10⁹ of l-EGFP phage (containing 250 ng DNA) and exhibited specific anti-EGFP responses 28 days post-vaccination. Rabbits were vaccinated i.m. with 4x10¹⁰ of l-HBsAg phage (2 microg DNA) or recombinant HBsAg protein. Following 2 vaccinations with l-HBsAg, 1 out of 4 rabbits exhibited high level anti-HBsAg responses (comparable to those seen using the recombinant HBsAg protein). Following a third vaccination with l-HBsAg, all 4 rabbits showed similar high level responses which have not decreased after > 6 months. High anti-phage responses were observed in all animals following the first immunization with l-HBsAg, indicating that a high antibody titre against the phage carrier did not prevent a subsequent immune response against the DNA vaccine component. Compared to results in mice using equivalent l-HBsAg doses, anti-HBsAg responses were much higher in rabbits, which could indicate a swamping effect in mice. Since phage l DNA is approximately 50 kb in size (10-fold larger than most plasmid vectors used for naked DNA immunisation), a comparable dose of phage l DNA given as intact phage particles actually delivers 10-fold less vaccine DNA on a per gene copy (molar) basis. Thus the efficiency of the technique may be even higher than the data at first suggests^ref. Whole bacteriophage l particles, containing reporter genes under the control of the cytomegalovirus promoter (P(CMV)), have been used as delivery vehicles for nucleic acid immunisation. Following intramuscular injection of mice with l-gt11 containing the gene for hepatitis B surface antigen (HBsAg), anti-HBsAg responses in excess of 150 mIU/ml were detected. When isolated peritoneal macrophages were incubated with whole l particles containing the gene for green fluorescent protein (GFP) under the control of P(CMV), GFP antigen was detected on the macrophage surface 8 h later. Results suggested that direct targeting of antigen-presenting cells by bacteriophage 'vaccines' may occur, leading to enhanced immune responses compared to naked DNA delivery. Bacteriophage DNA vaccines offer several advantages: they do not contain antibiotic resistance genes, they offer a large cloning capacity (approximately 15 kb), the DNA is protected from environmental degradation, they offer the potential for oral delivery, and large-scale production is cheap, easy and extremely rapid^ref.
• heteroplexes / DNA-chemical carrier complexes : artificial virus-like multicomponent systems / particles. They can circumvent the current limiting factors of viral vectors such as immunogenicity, size limitations of the transgene, potential mutation to replication-competent recombinant viruses and the expensive production.
• plasmid DNA protected from nuclease degradation and with sustained dispersion control by water-soluble polymers (micro/nanoparticles)
• not biodegradable polymers
• poly-vinyl pyrrolidone (PVP)
• biodegradable polymers
• cationic
• poly[a-(4-aminobutyl)-L-glycolic acid] (PAGA) (CytoPure™); developed by Nitto Denko Corporation, sold by Qbiogene, Inc.) : it is a derivative of poly-L-Lys in which the ester link is substituted with amide. Once released from endosomes, PAGA enters nucleus and is degraded to its monomer, L-oxylysine, which would be rapidly removed from the cellular compartments followed by metabolism and excretion from the body, preventing it from eliciting any antigenic or inflammatory response on its own.
• poly(2-aminoethyl propylene phosphate) (PPE-EA)


M_r = 3.03 x 104, Mn = 1.82 x 104 , n = 100.2. The ultimate hydrolytic degradation products of this polymer are expected to be a-propylene glycol, phosphate and ethanolamine, all with minimal toxicity profiles. PPE-EA has a relatively higher M_r compared with most other biodegradable gene carrier : this feature coupled with the pedent chain charges increased the condensing ability of PPE-EA for DNA, so that complete binding of DNA is achieved with PPE-EA at a charge ratio of 1, which is several fold lower than other biodegradable gene carriers.
• polyanhydride : multifunctional anhydride monomers were photocrosslinked to produce hydrophobic, highly crosslinked polymer networks that degrade by surface erosion. Surface-eroding polymers can deliver molecules of a wide range of sizes at sustained, steady rates, which is advantageous for DNA delivery, where the high molecular weight may complicate control of the release profiles. When plasmid DNA was released from photocrosslinked polyanhydride matrices, DNA recovery was low (approximately 25%). Electrophoresis indicated that the plasmid DNA was released primarily in the relaxed and supercoiled forms, yet the relative fraction of released DNA in the supercoiled form decreased over time. To improve DNA recovery and reduce the damaging effects of polymer degradation, DNA was pre-encapsulated in alginate microparticles, which served as a temporary coating that quickly dissolved upon microparticle release from the polyanhydride matrix. As photocrosslinked polyanhydrides have highly predictable drug release profiles that depend on the polymer erosion rate and implant geometry and not on the entrapped molecule size, they can serve dual purposes in many biomaterial applications where structural support and drug release would be beneficial^ref.
• polyethylene oxide-succinimidyl glutarate^ref
• polyethylene oxide amine / poloxamine^{ref1, ref2}
• poly(lactide-co-phosphate)
• poly (D,L-lactide-co-glycolic acid) (PLGA) polymers undergo bulk hydrolysis, resulting in a dramatic drop in pH that can significantly degrade encapsulated DNA molecules, and their DNA release rate is difficult to regulate. They take too long to release encapsulated payload and fail to induce high levels of target gene expression^ref. Researchers have used either UV spectroscopy or fluorometry with PicoGreen((R)) dye to quantify PLG-encapsulated DNA. While the sensitivity of DNA detection and quantification by PicoGreen is higher (approximately 12 pg/ml) compared to UV (approximately 0.5 mg/ml), each method as an analytical tool has limitations^ref
• a blend matrix formed by a poly D,L-lactic-co-glycolic acid (PLGA) copolymer and polyoxyethylene derivatives. More specifically, nanostructures with different PLGA:poloxamer and PLGA:poloxamine compositions were prepared by an optimized emulsification-solvent diffusion technique and studied the potential of these carriers for the encapsulation and controlled release of plasmid DNA. Depending on the particle composition, the encapsulation efficiency of the model plasmid pEGFP-C1 varied between 30% and 45%. All formulations provided continuous and controlled release of the plasmid with minimal burst effect. In addition, the release rate and duration was dependent on the composition of the particle matrix. Moreover, gel electrophoresis and cell culture (MCF-7 cell line) assays allowed us to confirm that the biologically active form of the plasmid was preserved during the particle preparation process and also during its release. Cell culture experiments also indicated that the new nanoparticles do not exhibit toxic effects on these cells at concentrations up to 5 mg/mL. Altogether, these results indicate that these composite nanostructures present a promising approach for the delivery of plasmid DNA^ref.
• chemically cross-linked hydrogels composed of Pluronic^TM, water-soluble tri-block copolymers of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), were synthesized by a photo-polymerization method to achieve controlled DNA release. Pluronic F127 was di-acrylated to form a macromer and cross-linked to form a hydrogel structure in the presence and absence of vinyl group-modified hyaluronic acid (HA). UV irradiation time and the presence of the vinyl group-modified HA affected the mechanical property of Pluronic hydrogels to a great extent. Swelling ratio, degradation, and rheological behaviors of Pluronic hydrogels were investigated. When plasmid DNA was loaded in the hydrogels for sustained delivery, various release profiles were attained by varying UV irradiation time and modified HA amounts. Entrapped DNA was gradually damaged with increasing the UV exposure time as evidenced by decreasing the transfection efficiency. The DNA fractions released from the HA/Pluronic hydrogels, however, exhibited considerable transfection efficiencies commensurate with the UV exposure time, suggesting that they were not chemically degraded during the release period and substantially maintained functional gene expression activities despite the UV irradiation^ref.
• poly(ortho esters) (POE) exhibit surface-restricted hydrolysis, resulting in a milder pH shift less likely to result in severe DNA degradation : POE-2 differs from POE-1 in the inclusion of a compound containing a tertiary amine group that can become positively charged in mildly acidic conditions, attracting DNA molecules and thus delaying their release. POE-1 and POE-2 readily form into microspheres of ~5 µm in diameter, capable of containing and protecting DNA molecules from degradation, and suitable for long-term storage. Both types of spheres hydrolyzed and released their DNA payload following acidification in vitro, but POE-2 samples took significantly longer for complete release. Although the acidic conditions resulted in some degradation of released DNA, encapsulated molecules retained their integrity. They are ideal for DNA vaccines, where the encapsulating material should degrade via hydrolysis rather than enzymatic mechanisms, and this breakdown should not release byproducts that may prove toxic to the host or harmful to the enclosed DNA^ref.
• degradable, pH-sensitive poly-b amino ester + poly D,L-lactic-co-glycolic acid (PLGA) generate an increase of 3–5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells^ref.
• polyfection, i.e. RME of polyplex (molecular conjugate made up of nonlipidic polycation bound by electrostatic interactions to DNA. They have low stability in serum. RME of the polyplex can be achieved if the DNA can be condensed to Ø < 100 nm, while access to tumour tissue from the vasculature is limited to particles with Ø < 70 nm. Complex Ø = 50-300 nm can generally be achieved, while careful choice of the polymer molecular weight can produce complexes with an average Ø < 30 nm. Careful control of mixing conditions and salt concentrations has been reported to produce complexes as small as Ø = 12 nm. Condensation is produced by neutralization of the charges on the DNA molecule, followed by hydrophobic collapse as water is displaced from the DNA structure. One of the major advantages of using polycations to condense DNA is that very large DNA molecules can be used (up to 45 Kb) : this allows the incorporation of gene regulatory regions such as LCRs that may afford better control of gene expression. The nonlipidic polycation (A) may be :
• cationic (ie L-Lys-containing) polypeptides
• synthetic peptides
• oligo-L-Lys
• poly-L-Lys (pLL). LD₅₀ to HEK cells < 40 mg/mL.
• lipopoly-L-Lys
• His/Lys (H-K) copolymer or histidylated poly-L-Lys : His become cationic upon protonation of the imidazole groups at slightly acidic pH. This protonated polymer induces the leakage of plasmid/hystidylated poly-L-Lys complexes from acidic vesicles and hence favors plasmid release into the cytosol.
• 16 Lys and an RGD peptide sequence (the latter facilitates binding of the peptide/DNA complex to the integrin receptor on Caco-2 cells in vitro)
• chromatin proteins
• protamine (sulfate)
• histone proteins
• human histone derived peptides
• Ser-Pro-Lys-Lys from the C-terminus of the histone H1 protein
• peptide Tyr-Lys-Ala-(Lys)₈-Trp-Lys
• m (mu) peptide (associated with the condensed core complex of Ad) : Met-Arg-Arg-Ala-His-His-Arg-Arg-Arg-

Arg-Ala-Ser-His-Arg-Arg-Met-Arg-Gly-Gly
• polyamines
• spermidine
• diethylaminoethyl (DEAE)

polyethylenimine (pEI) is a highly branched polymer composed of units which have 2 units of carbon per nitrogen with these units randomly distributed in the approximate ratios of 1 primary amino nitrogen:2 secondary amino nitrogens:1 tertiary amino nitrogen. When pEI is in dissolved in water the amino nitrogens react with the solvent to form positively charges nitrogens. Not all of the amino groups will become protonated, however, and the addition of acid will increase the number of charged amino groups. It has been suggested that only around 60% of the amino groups are charged in water, leaving 40% that can become protonated at lower pH, such as that which exists in the endosomes ("proton sponge hypothesis"). It has been suggested that this protonation in the endosome leads to endosomal swelling and bursting with subsequent enhancement of transfection activity. LD₅₀ to HEK cells < 20 mg/mL. PEI is a potent, but not harmless, mucosal immuno-stimulator of mixed T_h1/T_h2 response, activation of both CD8⁺ and CD4⁺ T cells, with a larger effect on CD4⁺, and FasL-mediated antigen-induced cell death
• EXGen500^® : linear 22 kDa pEI
• poly-dimethylammonio ethyl methacrylate (pDMAEM)
• poly-trimethylammonio ethyl methacrylate (pTMAEM)
• FuGENE 6^® : a proprietary blend of lipids (non-liposomal) and other compounds in 80% ethanol
• X-tremeGENE Q2^® : dried lipid film to rehydrate before use.
• lipofection : transfer of liposome and DNA complex (lipoplex)
• multilamellar large vesicles (MLV)

=> downsizing =>
• small unilamellar vesicles (SUV) : 50 ±10 nm

or
• large unilamellar vesicles (LUV) or liposomes (13% of all gene therapies) : no capacity limit ; low transfection efficacy due to rare fusion and migration through nuclear pores ; efficacy may be improved inserting ligand proteins in the bilayer ; very rare random integration in chromosomes.
• dendrimers : highly branched polymers made up of more generations (G) of a monomer
• PAMAM or PAA (poly(amidoamine))
• SuperFect (SF)^® : a mixture of branched polyamines derived from heat induced degradation of PAMAM dendrimer
• classical or anionic liposomes allow nucleic acid to enter the vesicle : there are problems with the efficiency of nucleic acid encapsulation, coupled with a requirement to separate the DNA-liposome complexes from "ghost" vesicles.
• cationic liposomes (CL) : cationic lipids are able to interact spontaneously with negatively charged DNA to form clusters of aggregated vesicles along the nucleic acid. At a critical liposome density the DNA is condensed and becomes encapsulated within a lipid bilayer, although there is also some evidence that cationic liposomes do not actually encapsulate the DNA, but instead bind along the surface of the DNA, maintaining its original size and shape. Cationic liposomes are also able to interact with polyanionic heparan sulfate proteoglycans (HSPG) on cell membranes more readily than classical liposomes. Fusion between cationic vesicles and cell surfaces might result in delivery of the DNA directly across the plasma membrane, bypassing the endosomal-lysosomal route which leads to degradation of anionic liposome formulations. Cationic liposomes can be formed from a variety of cationic lipids / cytofectins :
• cationic derivatives of cholesterol
• ACHx / CJE52 (3-aza-N¹-cholesteryloxycarbonylhexane-1,6-diamine)
• CDAN / B198 (N¹-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine)
• CHEMS (cholesteryl hemisuccinate)
• CTAP (N¹⁵-cholesteryloxycarbonyl-3,7,12-triazapentadecane-1,15-diamine)
• O-Chol (3b[L-ornithinamide-carbamoyl] cholesterol)


Note that the carbamoyl bond is readily degraded by abundant esterase within the cell, and the amide form of ornithine contains a positively charged terminal amine group.
• DC-chol (3a-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride)
• DC-Chol (3b-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol)
• quaternary ammonium compounds
• DDAB (dimethyldioctadecylammonium bromide)


• DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyl ethyl ammonium bromide)


• DODAC (N,N-dioleyl-N,N-dimethylammonium chloride)
• DOJMA (dioleyloxypropyl-3-trimethyl ammonium bromide)
• DOSPA (2'-(1",2"-dioleoyloxypropyldimethyl ammonium bromide)-N-ethyl-6-amidospermine tetratrifluoroacetic acid salt


• DOSPER (1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamid)


• DOTAP (N-1(-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium methylsulphate)


• DOTMA (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride or bromide)


• lipid derivatives of polyamines
• DOGS (Transfectam^®)


• lipofectAMINE Plus/2000
• cationic derivatives of diacylglycerol
• DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)
• methyl-b-destrin
• Tween-80
Cationic liposomes also usually incorporate a neutral lipid (helper lipid) into the formulation in order to facilitate membrane fusion :
• CHOL (cholesterol)
• DLPC (1,2-dilauroyl-sn-glycero-3-phosphatidylcholine)
• DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine)
• DOPE (1,2-dioleoyl-sn-glycero-3-L-a-phosphatidylethanolamine)


Such mixages are comercially available as :
• Lipofectin^® (DOTMA/DOPE 1:1 w:w)
• LipofectAMINE^® (DOSPA/DOPE 3:1 w:w)
• LipofectACE^® (DDAB/DOPE 1:2.5 w:w)
Cationic liposomes complexed with supercoiled plasmid DNA in solution self-assemble into a lamellar or an inverted hexagonal (H(I)) phase depending on lipid composition (cationic liposome / DNA complex). Particles that have not undergone maturation easily stimulate aggregation because of their dipole nature. Aggregated particles can become quite large (> 1500 nm) and are probably inactive for transfection. Fusion occurs because mixtures of cationic lipids and anionic phospholipids preferentially adopt the inverted hexagonal (H(II)) phase : cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.
• liposome / polycation / DNA (LPD) ternary complexes (lipopolyplexes) : at around 100 nm in size, most cationic liposomes are too large to pass through the tiny pores in the nuclear membrane except when the membrane breaks down during cell division. Even if cells are rapidly dividing, delivering genes via liposomes is not very efficient - and it is no good for slowly dividing cells such as those lining the lungs. Packaging with a polycation allows creation of liposomes 25 nm in size, small enough to enter the nuclear pores :
• liposome:mu:DNA (LMD)
• lipid:protamine:DNA (LPD)
These complexes showed higher transfection efficiency than the corresponding polycations-less liposome/DNA complexes, and also showed enhanced resistance to degradation by nucleases.
Markers for liposomes are non-exchangeable lipids labelled with fluorophores :
• 4-heptadecyl-7-hydroxycoumarin

fluorescein phosphatidylethanolamine (F-PE)
The potential biomedical utility of the photoinduced destabilization of liposomes depends in part on the use of green to near IR light with its inherent therapeutic advantages. The polymerization of bilayers can be sensitized to green light by adding to the lipid bilayer :
• amphiphilic cyanine dyes, i.e. the cationic 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI)
• the corresponding anionic disulfonated DiI (DiI-DS), with the lipid bilayer.
The DiI sensitization of the polymerization of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine /1,2-bis[10-(2', 4'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphocholine liposomes causes liposome destabilization, resulting in delivery of the contents of the endocytosed liposomes to the cytoplasm.
The liposome/DNA complexes used in clinical trials are directly cytotoxic in vitro : furthermore, studies in mice and macaques have demonstrated that exposure to high doses or to repeat doses of liposome/DNA complexes results in histopathology and gross lung pathology, suggesting that these vectors may not be as clinically safe as previously thought.
Pre-treatment with intravenous administration of GdCl₃ transiently depletes liver Kuppfer cells and spleen macrophages, preventing lipoplex uptake and subsequent cytokine production by these cells : such cytokines (TNF-a, IFN-gand IL-12) are otherwise toxic and inhibit transgene expression.
The transfection mechanism of the targeted cell can be described as a sequence of several transport and dismantling steps of the heteroplex :
• avoiding aggregation, nonspecific interactions with serum proteins, untargeted cells (endothelial cells, blood cells), phagocytosis, immunogenicity and non-specific cell uptake from anionic GAGs (heparan sulfate and hyaluronic acid). All these features can be achieved by incorporating a protective, interactive, non-condensing (PINC) polymer in the form of a neutral (non-ionic) hydrophilic shielding / coating :
• grafted co-polymers
• random co-polymers
• reactive hydrophilic polymers
• block co-polymers
• polyoxyethylene-polyoxypropylene (Pluronic 123)
• polyethylen glycol (PEG) ("stealth" liposomes)
• poly-N-2-hydroxypropyl methacrylamide (pHPMA)
• 1-vinyl-2-pyrrolidinone (VP)
• methylmethacrylate (MMA)
• Tf at high densities
... around polymer/DNA complexes, generating particles with a near neutral zeta potential.
• binding of the heteroplex to the cell surface and its subsequent internalization. It is possible to (covalently) add one or more functional groups (B), such as :
• ligand for cell receptor(s) ; C = ligand receptor(s) on the targeted cells.
• B = transferrin (Tf) ; C = CD71 / TfR (=> preferential targeting of proliferating cells, expecially hematopoietic cells). The number of transferrin molecules attached to each polylysine molecule varies according to the molecular weight of the polymer, but is generally around 1 transferrin molecule for every 50 lysine residues. The increase in density of the transferrin receptors on the surface of the cell that occurs following treatment of the cells with the iron chelator desferrioxamine.
• B = HBV L protein  ; C = (on hepatocytes)
• B = sugars or derivatives  (plasmid / glycosylated polycation complexes (glycoplexes)); C = lectins
• B = glycoproteins
• B = Gal-containing asialoglycoproteins (ASGP) (e.g. asialofetuin (A or B) or asialoorosomucoid (1 or 2)) ; C = Gal- and GalNAc-specific asialoglycoprotein receptor (ASGP-R) (1 or 2, on hepatocytes)
• B = small sugar units
• B = dextran ; C = ?
• B = Lac ; C = Gal- and GalNAc-specific asialoglycoprotein receptor (ASGP-R) (1 or 2, on hepatocytes). Lac allows low uptake but very efficient intracellular trafficking.
• B = a-D-Man pyranoside or Fuc; C = CD206 / macrophage Man receptor (MMR) : very efficient uptake, but delayed exit from endosomal compartment, high accumulation in lysosome, inefficient nuclear import and limited transcription of the complexed plasmid DNA.
• B = anti-CD3 Ab ; C = CD3deg
• B = invasin
• B = FGFs ; C = FGFRs
• B = VEGF ; C = VEGFR1 or VEGFR2
• B = cholera toxin B subunit ; C = GM₁
• B = folic acid ; C = folate receptor (FR) 1 (amplified in a wide variety of human tumors)

• B = polymeric IgA ; C = FcaRs
• B = Ad proteins ; C = coxsackievirus group B, adenovirus receptor (CVADR, CAR)
• B =  monoclonal antibodies or their Fab' fragments (immunoliposomes) ; C =  antigen
• type A : without PEG derivatives
• type B :
• type C : pendant-type PEG-immunoliposomes
However, one cannot exclude the possibility of direct entry into the cell by transient permeabilization of the cell membrane, expecially when heteroplexes containing liposomes or when amphiphilic peptides are used.
• endosomal release / escaping and cytoplasmic delivery
• treat cells with chloroquine (it is thought to have a buffering capacity that prevents endosomal acidification, leading to swelling and bursting of the endosomes and has been shown to enhance the transfection activity of polycation/DNA complexes). This approach is limited to in vitro applications since the concentrations of chloroquine required to enhance transfection are likely to be toxic in vivo.
• co-incubation of the cells with both adenovirus particles and heteroplexes. The use of Ad particles to enhance transfection of heteroplexes is unlikely to be widely used in vivo as the adenovirus particles may provoke inflammatory responses, while it has also been demonstrated that the transfection efficiency of these systems is much lower in vivo than in vitro.
• intrinsic components of heteroplexes
• His/Lys (H-K) copolymer or histidylated poly-L-Lys
• polyethylenimine (pEI)
• attachment of endosomolytic viral fusogenic peptides(=> fusogenic viral liposome)
• inactivated adenoviruses
• influenzavirus haemaglutinnin (HA). The fusion domain of this protein is located at the N-terminus of subunit HA 2 and this peptide sequence, and modifications of it, have been shown to significantly enhance transfection efficiency in a number of heteroplex systems.
• HVJ (Parainfluenzavirus 1 (Sendai virus)) envelope fusion proteins
• endosomolytic lipids
• poly(propylacrylic acid) (PPAA)
• poly-L-glutamate inihibits microtubule assembly by interacting with microtubule assembly protein, preventing plasmid delivery to lysosomes, thereby increasing the amount of plasmid that enters the nucleus. Once in the nucleus, through its interaction with nuclear proteins such as histones, poly-L-glutamate may enhance the stability and subsequently retention of DNA in the nucleus.
• poly-L-aspartate
• poly-acrylic acid
Reduction-oxidation (redox) sensitive polymers have been exploited to control cytosolic DNA release triggered by disulfide bond reduction by intracellular glutathione:
• alkylated ornithinyl Cys derivative
• peptides containing a Cys residue and a continuous sequence of Lys residues
• reaching gene expression machinery
• developing a cytoplasmic expression system based on the Enterobacteria phage 7 (T7) RNA polymerase
• transport into the nucleus : the cytoskeleton hinders diffusion of particles larger than 10 nm (cytoplasmic entrapment). In the cytoplasm, Ca²⁺-sensitive nucleases restrict the half-life of plasmid DNA to 50-90'. Most proteins are ubiquinated and subsequently enters the Ub proteasome-dependent degradation pathway : proteasome inhibitors can enhance their half-life.
• through the dissociated nuclear envelope during mitosis (effective only in dividing cells !)
• through the nuclear pore complex (NPC) : it is thought that less than 1% of the plasmid molecules that reach the cytoplasm eventually reach the nucleus.
• intrinsic components of heteroplexes
• lactosylated poly-L-Lys (pLL)
• certain sequences of the plasmid DNA recognized by transcription factors
• attachment of extrinsic nuclear localisation signal (NLS) from karyophilic proteins to the heteroplex :
• SV40 large T antigen (NLS^T : ¹²⁶PKKKRKV¹³²)
• D-NLS^T : D-stereoisomer
• mNLS^T : mutated NLS^T deficient on facilitated nuclear transport (used as negative control)
• revNLS^T : reversed NLS^T deficient on facilitated nuclear transport (used as negative control)
• Loligomer4 : Lys-based branched multidomain peptide containing 8 NLS^Ts.
• MPG : bifunctional peptide containing NLS^T and a hydrophobic fusion sequence from HIV-1 gp41.
• NLS^T together with its natural flanking regions, containing a Ser phosphorylation site which is recognized by CK2. Phosphorylation of this site is known to enhance binding of the NLS to its receptor importin a/b and consequently its rate of nuclear import.
• NL-NLS
• P101
• P101T
• M9 from hnRNP A1 (a "nonclassical" NLS rich in Gly and aromatic residues : ²⁶⁸NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY³⁰⁵)
• scrambled sequence of M9 (scM9) (used as negative control)
• H1 histone family members
• Vpr from HIV-1 (⁵²DTWTGVEALIRILQQLLFIHFRIGCRHSRIGIIQQRRTRNGA⁹³). The originality of this NLS relies on its ability to mediate nuclear import of a reporter protein independent of the importin a/b or transportin pathway, probably through direct interaction with the NPC and without the requirement of energy.
• fiber protein from Ad3 (peptide I : ¹AKRARLSTSFNPVYPYEDES²⁰) : this peptide contains a hydrophobic domain (FNPVYPY), a NPXY domain capable or RME and NLS (²KRAR⁵).
• HMG proteins
• fusogenic peptide INF7
• adenovirus hexon protein
The NLS can be attached to the DNA in the heteroplex ...
• covalently : care has to be taken to avoid binding of the NLS within the expression cassette blocking subsequent transcription of the transgene.
• lissamine
• cyclopropapyrroloindole (CPI)
• noncovalently (electrostatic interaction with the negatively charged DNA)
• by taking advantage of cationic residues within the NLS itself
• by fusion or covalent binding of a cationic moiety (poly/oligoLys, histone 1-DNA-binding domain, scrambled NLS^T) to the NLS.
• peptide nucleic acid (PNA) sequence, which functions as a sequence-specific DNA-binding domain. This allows control of both the number of attached molecules and their binding site on the DNA.
• oligodeoxynucleotide (ODN) covalently attached to the target DNA sequence using photo-activated psoralen (Pso-ODN)
• streptavidin-biotinylated DNA
Anyway it should be noted that the charge ratio of the complex and the chosen transfection system readily determine the maximum transfection level and are able to surpass the effect of the NLS. Furthermore some nonspecific functions of the NLS during transfection are possible : cationic residues could interact with heparan sulfate proteoglycans (HSPG) on cell membrane and nucleus and enhance cell entry of the heteroplex and possibly interfere with other steps of the gene transfer mechanism, while anionic residues could also interact with the polycations typically used for DNA condensation or the hydrophobic residues with lipid components.
• plasmids in conjugating Bacteria
• replication-defective viral vectors (non-essential genes are provided in trans by an helper virus, a plasmid or in the genome of the packaging cell line ; vector-producing cells (VPC) allow amplification ; they all induce immune responses; relatively small capacity for therapeutic DNA ; safety concerns (replication-proficient viruses (RPVs)) ; tropism of enveloped viruses can be modified by adding specific envelope glycoproteins (pseudotyping).
• dsDNA viruses, no RNA stage
• Adenoviridae (used in 28% of clinical protocols)
• Atadenovirus : Ovine adenovirus (OAd) : they use an uncharacterized receptor, can bypass pre-existing immunity to human adenoviruses but induce an immune response when given intravenously.
• Mastadenovirus : Human adenovirus (Ad) serotypes A, C, D, E, and F (26% of all gene therapies) : capacity 30 Kbp; quite stable, manipulation friendly genome; max [particles] > 10¹¹ / mL; they use 2 receptors:
• primary receptor : Coxsackievirus B and Adenovirus receptor (CAR / CVADR / CXADR), which interacts with the head domain of the adenoviral fiber protein, also called fiber knob. CAR is almost ubiquitously expressed (expecially in hepatocytes and in basolateral surface or human airway epithelial cells; instead undergoes transient down-regulation in primary tumour and re-expression in metastases). Artificial extension of the fiber shaft length (which normally ranges from 6 to 23 b-repeats) inhibits CAR-dependent entry. After the initial binding step, the viral particle achieves endocytotic internalization via interaction with ...
• secondary receptors : a_vb₃, a_vb₅ (and a_vb₁?) integrins, which interact with the penton base. Artificial extension of the fiber shaft length in combination with HI loop ligands (eg RGD peptide ligand) augments infectivity in CAR^- cells without enhancing hepatotropism in vivo.
Deficiency of the primary and/or secondary receptors in tumor cells causes relative resistance of target tissues to adenovirus-based gene therapy. Very high infection efficacy in vitro; in vivo, following intravenous injection, 90% of the administered vector is degraded in the liver by a non-immune mediated mechanism. Uptake by those cell types that don't express CAR (eg DCs) can be enhanced by ..
• bispecific antibodies targeting both Ad surface proteins and host cell molecules. E.g. 9B9 to target CD143 / angiotensin-converting enzyme (ACE) / kininase II in pulmonary endothelial cells.
• chimeric bridges consisting of the extracellular portion of CAR and cell-specific ligands (including specific mAb or scFv).
The virion migrates to the nucleus where genome remains as episome. VA-1 ribozyme increases translation rate, including translation of the transgene. Most commonly used strains are :
• subgroup B
• serotype 35 (Ad35) shows efficient gene transfer into human bone marrow CD34⁺ cells and human HSCs/progenitors^ref
• subgroup C
• serotype 2 (Ad2)
• setotype 5 (Ad5) : upon local delivery, Ad5 viruses use the coxsackie and adenovirus receptor (CAR) for cell binding and  integrins for internalisation. When administered systemically, however, their role in liver tropism is limited since CAR-permissive and mutated viruses show similar biodistribution, a finding recently attributed to blood coagulation factor IX or complement protein C4BP binding to the adenovirus fiber and " bridging" to either low-density lipoprotein receptor-related protein or heparan sulphate proteoglycans. Hepatocyte transduction in vitro can be enhanced by the vitamin K-dependent factors FX, protein C and FVII in addition to FIX, but not by FII, FXI and FXII. This phenomenon was not dependent on proteolytic activation or cell signalling activity and for FX was mediated by direct virus:factor binding. Human FX substantially enhanced hepatocyte transduction by CAR-permissive and mutated viruses in an ex vivo liver perfusion model. In vivo, global downregulation of vitamin K-dependent zymogens by warfarin significantly diminished liver uptake of CAR-deleted adenoviruses, however this phenomenon was fully rescued by acute infusion of human FX. Our results indicate a common and pivotal role for distinct vitamin K-dependent coagulation factors in mediating hepatocyte transduction by adenoviruses in vitro and in vivo^ref
Heavy (often secondary !) immune and inflammatory responses => short in vivo expression. A surprisingly high number of tetramer⁺ CD8⁺ T cells were found in the liver and lung and reached peak values at days 8 and 10, respectively, post-infection. Nearly 100% of these tetramer⁺ CD8⁺ T cells expressed high levels of granzyme B and IFN. Remarkably, liver viral load and liver enzyme elevation peaked early, at days 2 and 4, respectively, post-infection, before the specific CTL response was detectable. After generation of CTLs, there was only minimal liver damage or further decrease in virus titer. These results indicated that the primary peak response of tetramer⁺ CTLs does not correlate with the elimination of adenovirus or liver cytotoxic response^ref.
Virus immunogenicity may be decreased by :
• polyethylen glycol (PEG) ("stealth" viruses)
• helper-independent first-generation Ad vectors (FGAd) : generated by double crossing over event between 2 plasmids => E1A and E1B delted (=> capacity < 6 Kbp); blocking of virus genetic program can be leaky (=> cytopathic effect); high titre production levels (up to 10¹¹-10¹² PFUs/mL); high level expression of transgene in transduced cells. 15 days of survival in the host. This kind was used in therapy for cystic fibrosis (CF) (aerosol-transfected pneumocytes) and ornithine transcarbamylase (OTC) deficiency. In liver-directed gene therapy, FGAd lead to high but transient levels of transgene expression and long-term hepatotoxicity due to viral gene expression from the vector backbone. Furthermore, high doses also result in an acute innate inflammatory response with potentially lethal consequences.
• helper-independent 2^nd generation vectors :
• E1 deletion and ts E2A^ref
• E1 and E4 deletion (=> capacity < 9 Kbp) : on September 17, 2000, in a phase I trial led by James M.Wilson, infusion into the right hepatic artery of 6x10¹¹ particles/kg (3.8x10¹³) of human adenovirus type 5, deleted in E1 and E4, containing human OTC cDNA caused death of 18-year-old Jesse Gelsinger : approximately 18 h. following gene transfer the subject was noted to have altered mental status and jaundice--clinical signs not seen in any of the first 17 subjects in this study. Subsequently, his clinical course was marked by SIRS, biochemically detectable DIC, and MOF failure, leading to death 98 h following gene transfer. Post-mortem examination was consistent with the clinical course, and vector DNA sequences were readily detectable in most tissues. The subject had high serum levels of IL-6 and IL-10 but normal TNF-a immediately after infusion of the vector^ref.
• E1, E2A and E3 deletion
• E1, E2B and E3 deletion
• E1, E3, and E4 deletion
• E1 and E2B deletion^ref : reduce liver toxicities
=>  profound blocking of viral gene expression =>
• expression of transgene impaired
• reduced vector-induced cytopathic effect (CPE)
• vector persists longer (up to 6 months) in transduced cells
• helper-dependent (HD) Ad vectors (HDAd), "gutless" or "gutted" vectors (29.6 kb) depend on a helper virus (35.3 kb) for replication and contain only ITRs and packaging sequences apart from the transgene. Helper viruses are constructed with packaging signals flanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal is efficiently excised, rendering the helper virus genome unpackageable. However, the helper virus DNA is replicated at normal levels and can thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements. Serial passage of the vector in helper virus-infected 293Cre cells result in an approximately 10-fold increase in vector titer per passage. The vector can be partially separated from residual helper virus by CsCl buoyant density centrifugation. Large scale preparations of vector yield semi-purified stocks of approximately 10¹⁰ transducing virions/mL, with < 0.01% contamination by the E1-deleted helper virus (low but significant). Thisis a error-prone not robust production system (susceptible to recombiation and instability). This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity. They have higher levels and prolonged transgene expression. Anyway they lose efficiency in transduction and immune protection^ref. One concern is the occurrence of replication-competent adenovirus (RCA) and defective viral particle (1:10 or 1:200 ratio) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. There are massive scale production restrictions for clinical use due to purification restrictions. Unlike FGAd, HDAd contain no viral genes and can provide sustained, high-level transgene expression with negligible long-term toxicity : anyway systemic delivery of HDAd, like FGAd, results in acute toxicity in baboons consistent with activation of the innate inflammatory response, the severity of which is dose dependent, and confirm the hypothesis that Ad-mediated acute toxicity is independent of viral gene expression^ref.
• mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens have proved highly immunogenic in preclinical studies but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world. The 7 short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing SIV Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs^ref.
Production method : packaging cell line (HEK-293 : subclone D has high adenoviral productivity linked to integrin a_vb₃-mediated survival signaling events (via p44/42^MAPK)  that delay stress signals thus allowing more Ad production by the infected cell). Among the wide variety of methods used for the construction of recombinant AdVs, several allow for the attainment of recombinant viruses withour any background of parental genome. One widely used method to generate AdVs involves the co-transfection of restriction enzyme (ClaI) digested deproteinized DNA comprising most viral genes and the right ITR with a transfer vector containing the desired expression cassette, the left ITR, and a segment of Ad sequences common to both molecules which permits HR. In typical experiments, co-transfection of 1 mg of naked viral DNA with the same amount of transfer vector using ...
• calcium phosphate co-precipitation generates ~ 10÷30 recombinant plaques / mg of viral DNA, with an efficiency of 20÷60% of recombinants : this is not enough for generation of an Ad library.
• viral DNA-protein complex (DNA-TPC) generates 3500-5000 recombinant plaques / mg of viral DNA, with an efficiency of 60% of recombinants.
Thus, even though it is currently possible to generate several thousand viruses per mg of viral DNA, only a fraction of those will be recombinant : to minimize the work involved in screening recombinants from parental viruses, reporter genes can be used either in the viral genome as a negative screen, or in the transfer vector as a positive screen. Although useful, this approach still suffers from the intrinsic limitation that, in a library of several thousand clones, an even larger number of parental viruses would have to been screened out, a process which is fairly time-consuming. Furthermore, recombinant AdVs are sometimes at a growth disadvantage relative to the parental virus and these clones might be difficult to isolate in a library in which they are not positively selected for growth. One possible positive selection system compatible with the generation of very large numbers of AdV clones would be to ectopically re-express an essential genes in such a way that only those viral genomes having incorporated this gene would be able to grow in the selective environment. The Ad protease (PS) gene is a good candidate for such a purpose : it is one of the essential late viral genes involved in many steps of the virus cycle. PS-deleted Ad mutants with or without E1 and E3 deletions can be propagated in HEK-293 cells transfected with a plasmid harboring the left ITR, a PS-expressing cassette with or without a foreign gene, and a region allowing for HR in E1. Such a cell line expresses PS under the control of an enhancerless drug-inducible promoter : the expression of the gene of interest is not only unnecessary, but also potentially deleterious during recombinant AdV generation and production; furthermore, regulation of transgene expression is essential to ensure proper representation of every insert in a library. Because the PS gene is not required for DNA replication, the DPS mutants are capable of a single round of replication when E1 functions are provided in trans as in 293 cells, so that the parental Ad can be eliminated after one round of replication, while those genomes having rescued the PS gene can lead to productive infection.
Use of column chromatographic steps instead of CsCl gradient purification greatly eased process scale-up.
Transcriptional interference mediated by several cis-elements, mainly located on the left end of the adenovirus chromosome, appears to be accountable for loss of tissue specificity or down-regulation of promoters used to drive transgene expression. The ITR contains redundant binding sites for cellular factors and stimulates transcription from E1 and E4 genes. The bipartite E1A enhancer trans-activates the E1A gene through  the cellular protein EF-1A and controls the expression of the early genes after infection. The promoter for the intermediate transcript pIX is situated just downstream of the canonical E1 cloning site for expression cassettes. Since some of these elements overlap with the viral origin of replication and the packaging signal, they cannot be deleted or inactivated. Thus other strategies based on the use of transcription modulators have been developed to achieve targeted gene delivery ... :
• transcriptional terminator from bovine or human growth hormone
• transcriptional insulator elements from the chicken g-globin locus (HS-4)
... but they are subject to important functional limitations. Loss of tissue-specific expression for transgenes cloned in the rightward-orientation could occur by a passive readthrough of the heterologous promoter, rather than by an active and direct activation of it from 5' viral enhancers.
• Human adenovirus serotype B uses CD46 / MCP as a receptor : they can transduce hematopoietic stem cells, dendritic cells and malignant tumor cells, that are refractory to infection by commonly used adenoviral vectors
• Baculoviridae : Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) : the virus enters insect cells via unidentified receptor-mediated endocytosis. Baculoviruses also transduce both dividing and nondividing mammalian cells, are nonreplicative in mammalian cells, nonpathogenic in humans and only low levels of systemic anti-baculovirus antibodies have been detects. They specifically transduce cuboid epithelium of the choroid plexus in ventricles or rat brains.
• Herpesviridae (1% of all gene therapies) : Alphaherpesvirinae
• HHV-1 / HSV-1 : capacity 20 Kbp ; neurotropic ; up to 20% of experimented animals may die shortly after injection.
• amplicons : bacterially produced plasmids containing ColE1 ori, OriS (the HSV-1 origin of replication), HSV-1 packaging sequence, the transgene under the control of an IE promoter (a4 or a22), a selectable marker, oriP element and EBNA1 gene from HHV-4 / EBV for plasmid episomal maintenance. Strong inflammatory responses.
• recombinant HSV-1 : replication-conditional mutants defective in one or more of the IEGs (HSV-tk, ribonucleotide reductase, UTPase, neurovirulence factor g34.5), which are provided in trans.
• disabled infectious single cycle (DISC) vectors : delection of HSV-tk and HSV envelope glycoprotein H (gH) allows 1 only replicative cycle to complete.
• replication incompetent ICP4-deleted HSV
• replication-defective derivatives of HSV-1 with deletions of the IEG ICP4, ICP22 and ICP27.
• HHV-2 / HSV-2
• recombinant HSV : replication-conditional mutants defective in one or more of the IEGs (HSV-tk, ribonucleotide reductase, UTPase, neurovirulence factor g34.5), which are provided in trans.
• disabled infectious single cycle (DISC) vectors : delection of HSV-tk and HSV envelope glycoprotein H (gH) allows 1 only replicative cycle to complete.
• herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency and the viral genome persists as high copy number, circular, nonintegrated episomes which segregate to progeny upon cell division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. The dissemination of HVS-based vectorsin vivo has been assessed following intravenous and intraperitoneal administration. Bioluminescence imaging of an HVS-based vector expressing luciferase demonstrates that the virus can infect and establish a persistent latent infection in a variety of mouse tissues. Moreover, the long-term in vivo maintenance of the HVS genome as a nonintegrated circular episome provided sustained expression of luciferase over a 10-week period. A particularly high level of transgene expression in the liver and the ability of HVS to infect and persist in hepatic stellate cells suggest that HVS-based vectors may have potential for the treatment of inherited and acquired liver diseases^ref
• Poxviridae (6% of all gene therapies) : Chordopoxvirinae
•  Avipoxvirus
• Canarypox virus : ALVAC recombinants have been administered to humans and mammalians by parenteral and oral routes showing abortive replication (block in the replication cycle in the human cell lines analyzed occurred prior to DNA replication), no systemic dissemination or severe reaction. Recombinants are immunogenic by the intramuscular and subcutaneous routes. They are also immunogenic when given orally, but the dose required is still under study. Canarypox recombinants effectively prime the immune system for induction of antibodies and CD8 cell-mediated cytotoxicity by protein antigens. Antibody responses are not influenced by prior inoculation of canarypox, of subunit vaccine corresponding to the gene insert, or of vaccinia. Canarypox virus is attenuated for canaries, in which species it is already widely used. In principle, it is non-infectious for humans or other mammals. It may be infectious for other birds.
• Orthopoxvirus
• Fowlpox virus (less immunoreactive than vaccinia viruses)
• Vaccinia virus
• Vaccinia virus (strain Ankara) / modified vaccinia Ankara (MVA)
• NYVAC
• ssDNA viruses : Parvoviridae : Parvovirinae : Dependovirus : adeno-associated virus (AAV) (2% of all gene therapies; weak immunogenicity due to few epitopes)


The traditional rAAV production system involves the transfection of ...
• AAV vector plasmid in which rep and cap are replaced by the poly-A containing transgene expression cassette (< 4.5 Kbp), demonstrating that only the ITRs are required in cis for replication and packaging of rAAV genomes
• AAV pDG helper plasmid containing the AAV rep and cap genes, MMTV LTR, some Ad genes (VA-1, E2A, E4 and RTR : other plasmids also contain E1A and E1B), but not ITRs
... by CaPO₄ precipitation (6 hrs) into 293 cells in presence of helper functions :
• superinfection from an helper virus
• Ad (E4-ORF 6)
• HHV-1 / HSV-1
• vaccinia virus
• physical agents :
• g-radiations
• X-rays
• UV
• heat shock
• genotoxic chemical agents => DSB => checkpoint induction : treatment with 3-AB causes increased PARP binding and lowered rAAV processing
• methyl methan sulfonate
• mitomycin C
• cisplatin
• DNA synthesis inhibitors, such as aphidicolin^ref or hydroxyurea^ref
• topoisomerase inhibitors^ref (novobiocin, etoposide, campthotecin)
• protease inhibitors
=> CPE for 48 hours => freeze/thaw and ammonium sulfate fractionation => CsCl₂ gradient centrifugation => collect and dialyze fractions => titration by QC-PCR => assay for transgene expression. Vectors retain < 10% of viral genome.
Progress has been made to increase rAAV yield through :
• potential increase in upstream production yields by using Ad gene plasmids in lieu of active Ad infection of packaging cells, and by moderating the amount of long Rep (Rep78 and Rep68) proteins expressed relative to short Reps (52/40) and capsid. Other improved production methods have included the use of stable cell lines with inducible Rep expression and rescuable vector genomes or the use of herpes simplex virus amplicons or recombinants. Recent purification strategies have sought to avoid CsCl ultracentrifugation, which seems to decrease the infectivity of vector material, and favoring column chromatography methods^{ref1, ref2, ref3, ref4, ref5}.
• regulating the AAV rep and cap gene expression
• eliminating the cytosolic helper virus
• using the SV40ori to increase AAV helper plasmid copy number.
Additional strategies to minimize the contamination of replication competent AAV (rcAAV) particles have also been proposed and studied, including :
• replacement of intact ITRs in the vector construct with a truncated D sequence (D10)
• splitting rep and cap sequences to different helper plasmids when making an AAV packaging cell line.
• replication competent AAV helper plasmid which retains the ITR (hRCAAV). In neither transfection-based systems nor systems with integrated AAV helper genomes can the copies of rep and cap genes synchronize to the replication of vector sequences. Although at the early stage of the wild-type AAV growth only a limited number of templates for transcription are available, it is sufficient to supply necessary Rep proteins for massive replication of the AAV genome. In the late stage of AAV growth, when large amounts of AAV Cap proteins are required for the packaging process, the already replicated AAV genomes allow the high level of cap expression to meet the demand of efficient encapsidation. In contrast, in the transfection-based rAAV production system, there are large numbers of AAV helper genomes available right after the transfection procedure. This results in the ovesupply of Rep proteins in the early stage of rAAV production. However, in the late stage of AAV packaging, AAV templates start to decrease because of cell division. This imbalance between rep and cap may contribute to the inefficiency of the current AAV production systems. Intronized AAV helpers containing AAV ITRs may achieve high efficiency rAAV packaging by mimicking wild-type AAV growth : the contamination of rcAAV is controlled by increasing the AAV helper genome through heterologous intron insertion (intronization) and utilizing an AAV mutant ITR that was defective in encapsidation, but still capable of supporting AAV replication.
Advantages :
• high titer stocks (max [particles] < 10^13÷14 / mL)
• limited host inflammation nor immune response (since they are devoid of any viral gene)
• long term expression (up to 4÷5 years)
• high level of expression
• infection of both dividing and non-dividing cells : permissivity to infection doesn't depend on expression of receptor, but rather on dsDNA formation, prevented in dividing cells
• specific tropism for
• skeletal
• smooth muscle cells
• cardiomyocytes
• neurons
• 5÷10% hepatocytes)
• in the skin they can transduce
• keratinocytes
• hair follicles
• sweat gland ducts
• panniculus carnosus (the skeletal muscle layer which in rodents is associated with the dermis)
• transduction of dendritic cells (DCs)
• the relative inefficiency of rAAV2-based gene delivery has led to studies of potential barriers to efficient gene transfer in tissues like lung^ref. The first of these barriers to be clearly identified was the relative paucity of the common AAV2 attachment receptor, heparan sulfate proteoglycan, on the apical surface of polarized ciliated respiratory epithelial cells. Comparison with vectors packaged in AAV5-based capsids, which utilize N-linked sialic acid as an attachment receptor indicated that this receptor abundance issue could be a major limiting factor to the efficiency of airway epithelial cell transduction^{ref1, ref2}. The potential utility of the newly available pseudotyped rAAV vectors for improved transduction of other organs, such as liver and muscle have also been studied^{ref1, ref2}. These tend to indicate a significant advantage of rAAV1 vectors for muscle transduction and of rAAV6 in the liver.
Importantly, it has been demonstrated in vitro that viral tropism can be changed by inserting ligands specific for relatively resistant cells into the capsid protein. The targeting of receptors such as the serpin enzyme complex receptor^ref, the LDL-receptor^ref, and several others^ref have been successful. Even in tissues, like skeletal muscle, where rAAV2-based vector transduction appears to be relatively robust, substantial increases in vector transduction efficiency can be observed by the use of alternative serotype capsids. For instance, an improvement of transduction efficiency with rAAV2-based vectors of several-hundred fold was observed in factor IX and AAT vectors crosspackaged into rAAV1 capsids^ref. Similar enhancement of transduction efficiency has also been seen in the central nervous system with other serotypes, although in that case, the pattern of cell types transduced also differs based on which serotype capsid was used^ref.
• as rAAVs lack rep, they can't integrate. Infection with AAV vectors do not increase mutation rates in normal human cells. In hepatocytes, although stable transgene expression primarily results from extrachromosomal vector genomes, a series of experiments has shown that vector genomes integrate into host chromosomes in hepatocytes at a low frequency. Despite the low integration efficiency, recent reports of retroviral insertional mutagenesis in mice and 2 human subjects have raised concerns about the potential for rAAV2-mediated insertional mutagenesis. Frequent chromosomal deletions of up to 2 kb at integration sites (100%; most of the deletions are <0.3 kb) and preferred integration into genes (72%). In addition, all of the targeted genes analyzed (100%) were expressed in the liver.
• expression of the therapeutic gene can be driven by any desirable promoter
• cells are tranduced at high multiplicity of infection (MOI) : different rAAV preparations can be mixed to obtain simultaneous expression of appropriate gene combinations in vivo. rAAV-mediated gene expression can be enhanced through intermolecular cis activation by using a dual-vector approach : 2 independent rAAV viruses, one encoding a transgene with or without a minimal promoter and another with enhancer sequences are used to co-infect the same tissue. Subsequent intermolecular concatamerization between 2 rAAV vectors substantially augments expression of the transgene because of the presence of enhancer elements within the same circularized molecule^ref.
Problems :
• limited cloning capacity : even in presence of genotoxic agents, most of the vectors remains in endosomal compartments and cannot enter the nucleus
• extablishment of highly efficient rep-expressing packaging cell lines is often a challenge and very labor-intensive as AAV Rep proteins are highly cytoxic when overexpressed in mammalian cells
• helper functions
• receptors :
• heparan sulfate proteoglycan (HSPG) is poorly expressed on the apical surface of polarized ciliated respiratory epithelial cells.
• AAV1 efficiently transduces both skeletal and cardiac muscles^ref
• AAV2 :
• the broad host range might represent a limitation for some applications in vivo, because rAAV-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid protein, thereby redirecting the binding of AAV2 to other cellular receptors^ref.
• levels of transduction of the vascular endothelium by AAV-2 are low, an observation linked to deficiencies in endothelial cell binding, sequestration of virions in the extracellular matrix and/or virion degradation by the proteasome. Strategies to improve transduction of endothelial cells include AAV-2 capsid targeting using small peptides isolated by phage display or the use of alternate serotypes
• AAV3 transduces endothelial cells with poor efficiency
• AAV4 targets ependyma and astrocytes in the subventricular zone and rostral migratory stream (RMS)^ref, but transduces endothelial cells with poor efficiency
• AAV5 transduces endothelial cells with poor efficiency. Immunization involving a DNA vaccine prime followed by an adenovirus type 5 (Ad5) boost elicited a protective immune response against SHIV challenge in monkeys. However, the hepatocellular tropism of Ad5 limits the safety of this viral vector. A replication-defective chimeric Ad5 vector with the Ad35 fiber (Ad5/35) showed minimal hepatotoxicity after intramuscular administration in BALB/c mice and rhesus monkey. In addition, an Ad5/35 vector expressing HIV Env gp160 protein (Ad5/35-HIV) generated strong HIV-specific immune responses in both animal models. Priming with a DNA vaccine followed by Ad5/35-HIV boosting yielded protection against a gp160-expressing vaccinia virus challenge in BALB/c mice. The Ad5/35-HIV vector was significantly less susceptible to the pre-existing Ad5 immunity than a comparable Ad5 vector. These findings indicate that an Ad5/35 vector-based HIV vaccine may be of considerable value for clinical use^ref.
• AAV6 transduces endothelial cells with poor efficiency
• AAV7 has been shown to mediate efficient transduction of the skeletal muscle but has poor efficiency at transducing endothelial cells due to proteasome degradation^ref
• AAV8 has been shown to mediate efficient transduction of the liver but has poor efficiency at transducing endothelial cells due to proteasome degradation^ref
• techniques allowing for the incorportation of peptides into capsid proteins of viral vectors opened the door to the possibility of exploiting receptor-specific targeting, while retaining the efficiency and stability conferred by the viral vector^{ref1, ref2, ref3} : at issue is the fact that the number of known ligands small enough for such a strategy is quite limited for some cell types. Incorporation of an Arg-Gly-Asp (RGD)-containing peptide at these sites enables AAV to infect integrin-expressing cells independent of HSPG^ref. Random small (7-mer) peptides can be incorporated within the heparan-binding loop on the exterior surface of the AAV capsid creating a diverse library of receptor-targeted rAAV constructs to screen for their ability to infect a specific cell type (e.g. SIGYPLP^ref for endothelial cells and other heptapeptides^ref for coronary endothelial cells, a target not very permissive for wt-AAV2, as are HSCs). The role of targeting is important even for cell types thatt are permissive for transduction by existing rAAV serotpyes, but whose regimen are poorly selective, e.g. in GDEPT or therapies currently causing germline gene transfer (e.g. rAAV2 genomes appear in the semen of male hemophiliacs receiving hepatic delivery of a rAAV2-factor IX vector).
• postentry limitations : subsequent studies have indicated that mechanisms beyond cell-surface attachment may account for some of the serotype-specific differences in gene transfer efficiency. In particular, rAAV2-based vectors when exposed to the apical surface of polarized airway epithelial cells appear to be capable of entering the cells with reasonably high efficiency^ref. However, the efficiency of nuclear entry is quite poor^{ref1, ref2} and can be enhanced by inhibition of proteosome function^ref. Paradoxically, ubiquitination appears to be required for entry into the nucleus by this pathway^ref. This implies a complex series  of trafficking signals necessary for vector to be transported to the nucleus, and these events may differ based on the cell type or polarity of vector entry into the cell^{ref1, ref2}. In any case, the newly widened availability of alternate AAV capsid serotypes and receptor-specific mutants promises to provide means for bypassing the various obstacles to vector entry. Once within the nucleus, the vector DNA must be converted from single-stranded DNA to double-stranded DNA in order to be transcriptionally active, a potentially inefficient process. The mechanism by which this usually occurs appears to be leading strand synthesis. This process appears to be regulated by the phosphorylation state of a single-stranded d-sequence binding protein, which has been identified to be a member of the FKBP family^ref. One way around this particular problem has recently been described. This method involves the design of vectors that represent self-complementary doublets of the sequence of interest, capable of folding back upon themselves upon uncoating of the virus to produce a vector genome that very rapidly becomes transcriptionally active^ref.
• stable persistence :
• in the case of quiescent cells, Rep-deleted AAV vectors have demonstrated a propensity to form stable concatemers, apparently by nonhomologous end-joining (NHEJ) of vector monomer genomes^ref. Incidentally, this mechanism may also be taken advantage of to overcome the inherent packaging limit of AAV, by packaging 2 half molecules with splice donor and splice acceptor sites into 2 different populations of vector particles. Once these particles coinfect a target cell, concatemer formation will allow for the production of a full-length active vector^{ref1, ref2, ref3}
• in the case of proliferating cells, the lack of efficient integration in the absence of Rep may be problematic. Several groups have demonstrated that it may be feasible to add Rep back in trans in order to effect site-specific integration. The efficiency of this process appears to be lower than that seen for wild-type virus, however, suggesting that cis-acting sequences outside of the ITRs proper are required for efficient site-specific integration.
• a unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the ITRs of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of 26 cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of 5 of the cell lines by chromosme FISH confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors^ref.
A directed evolution approach involving the generation of large mutant capsid libraries and selection of AAV2 variants with enhanced properties has been performed. High-throughput selection processes were designed to isolate mutants within the library with altered affinities for heparin or the ability to evade antibody neutralization and deliver genes more efficiently than wild-type capsid in the presence of anti-AAV serum. This approach, which can be extended to additional gene delivery challenges and serotypes, directs viral evolution to generate 'designer' gene delivery vectors with specified, enhanced properties^ref.
Applications : the availability of high-titer, highly purified rAAV2 vector preparations, free of rcAAV has allowed for testing of rAAV2 gene delivery in numerous in vivo settings, including many rodent and nonhuman primate models. rAAV gene delivery was demonstrated in the lungs of rabbits, mice, and rhesus macaques in the context of the development of rAAV2-CFTR vectors for the treatment of cystic fibrosis (CF)^ref. These studies indicated that rAAV2 gene delivery to the lung was feasible, safe and persistent. Surprisingly, persistence was associated with stable episomal forms of the vector genome, rather than with integration. Indeed, the crucial role of the rAAV Rep protein (which is deleted from most rAAV2 vectors) in driving integration has since been confirmed. Subsequent studies showed that rAAV2 gene delivery was much more efficient in terminally differentiated neurons, retinal cells^ref, and myofibers. The delivery of rAAV2 vectors to the liver has also been demonstrated to be very efficient^{ref1, ref2, ref3, ref4, ref5, ref6}. rAAV2-based gene delivery to muscle and liver has been shown to be quite effective for delivery of secreted proteins, such as factor IX and a₁ antitrypsin^{ref1, ref2}. They have been used in protocols for A type hemophilia and B type hemophilia, mucopolysaccharidosis type VII, Fabry disease, Pompe disease, a canine model of  Leber congenital amaurosis, and Parkinson's disease (PD). The use of rAAV2 in early-phase clinical trials has been reported for 2 diseases, cystic fibrosis (CF) and hemophilia B. In the case of CF, 3 phase I trials and two phase II trials have been performed^{ref1, ref2, ref3}. The phase I trials have included delivery to the nose, maxillary sinus, and lung, with the latter organ approached both by direct bronchoscopic instillation and by aerosol inhalation. Phase II studies of maxillary sinus and aerosol administration have also been completed recently. Taken together, these studies generally indicate a lack of any vector-mediated toxicity, and a surprisingly efficient rate of DNA transfer. The phase II aerosol trial also showed evidence of a decrease in pulmonary inflammation and an increase in pulmonary function, both of which were statistically significant in this randomized, placebo-controlled, double-blinded study. The effects lasted only for 30 days, however, and repeated administration did not appear to be capable of sustaining the positive effects. This appears to relate to the development of neutralizing antibodies to AAV2 capsid components. The studies in patients with hemophilia B have yielded somewhat similar results^{ref1, ref2}. Gene transfer has been documented, and transient evidence of biological activity has been demonstrated. However, the efficiency and persistence of gene delivery does not appear to be sufficient for long-term therapeutic efficacy so far. In the case of hemophilia, it appears that immune responses to the transgene product, as well as the vector capsid may be the cause of some of the limitation, particularly when vector is administered by the intramuscular route. This could relate to the potential for factor IX binding the extracellular matrix in muscle, as has been demonstrated in factor IX-deficient mice. Because of this concern, the focus of trials has recently moved from intramuscular to intrahepatic administration. Although very few subjects have been enrolled to date, reports of transient presence of vector DNA sequences in the semen of male subjects has been a cause for increased scrutiny in these particular trials. As with the animal studies, the clinical trials have brought into focus a number of potential limitations to the rAAV vector platform. The limitations include a number of potential barriers to efficient entry, the lack of stable persistence of vector DNA within cell targets that are proliferating, which limits persistence, and the immune response to the vector capsid, which limits readministration. Finally, there remains the inherent limitation due to the constrained packaging capacity of the AAV capsid (5 kb total).
The potential limitations to rAAV-based gene delivery can be considered in the context of the steps that must be accomplished before efficient, persistent gene transfer and expression can occur. The first potential obstacle is efficient delivery and distribution to the appropriate cell surface. For some cells, such as the airway epithelial cells lining the airways of CF patients with pre-existing lung disease, a complex mixture of cellular and bacterial products is present within the airway lumen potentially providing an important extracellular barrier to gene transfer^ref, although the significance of this effect has recently been called into question^ref. Another important potential extracellular blockade to cellular attachment could be posed by the presence of extracellular neutralizing anti-capsid antibodies. Interestingly, clinical studies have not shown that pre-existing anti-AAV antibodies form a significant barrier to vector entry. However, repeated dosing and its associated rise in anti-AAV antibodies do appear to prevent efficient readministration, particularly in the respiratory tract. Although secreted IgA antibody has not been demonstrated, it does appear that compartmentalized immune responses may play a role. Another potential impediment to efficient delivery of vector to the target cell may occur when there is only a very brief time of contact between vector and host cell. This most prominently may occur within the lumen of vascular structures or airways, where vector may be swept along in the current of intralumenal fluid flow. The feasibility of surmounting this problem by reversibly binding vector to latex microspheres has recently been demonstrated^ref. The potential for repeated dosing of rAAV vectors has been studied in a number of preclinical models and utilizing a number of different modes of administration. In the case of airway surface administration, Beck et al reported that repeated administration of rAAV2 to rabbits by bronchoscopy led to the development of increasing titers of anti-AAV2 neutralizing antibodies. In that system, a third successive dose of rAAV vector yielded a lower (but not absent) efficiency of gene transfer, while an AAV3 pseudotyped vector was relatively unaffected. In a similar paradigm, Halbert et al reported a more dramatic decrement in efficiency of subsequent doses with rAAV2 vectors, an effect that could be abrogated by transient immunosuppression. However, they did not see as great an effect with rAAV6-based vectors^ref. Clinical trial data, like the animal data, indicate a consistent increase in serum neutralizing antibodies after dosing with rAAV2-CFTR, but inconsistent effect on blocking of gene transfer via the airway lumen. In all clinical trials, the level of pre-existing serum neutralizing antibodies was not predictive of gene transfer^{ref1, ref2, ref3,}. Taken in aggregate, these data suggest that the presence of serum neutralizing IgG antibodies may indicate that a humoral immune response to capsid components has occurred, but is not fully predictive of mucosal (most likely IgA-mediated immunity). If, as is generally the case, the IgA-mediated immunity is less persistent than the IgG-mediated immunity, a longer interval between doses may be indicated. Less data are available for intramuscular injection, but there again humoral responses to capsid have been observed. Unlike lung delivery, however, intramuscular gene transfer with rAAV has generally resulted in life-long effects, potentially obviating the repeated dosing issue.
Use of small molecules to overcome intracellular barriers to rAAV transduction : AAV vectors as a class appear to have the most important properties necessary for successful gene therapy, namely they are relatively nontoxic and are potentially capable of long-term persistence in human cells. A variety of obstacles to efficient gene delivery and persistence have been identified recently, allowing for more focused efforts to improve the technology. Fundamentally, none of these limitations appears to be intractable and early clinical trials show hopeful signs that the remarkable safety profile of this vector will hold true in its derivation as a human therapeutic agent. Most challenging will be the achievement of the final goal of life-long persistence, which based on the virus life cycle, does appear to be ultimately within reach. If this can be reproducibly achieved, the potential for curative therapy could follow close behind. One of the most immediate modifications that could come into clinical trial applications would be the use of small molecule proteasome inhibitors to enhance the delivery of rAAV vectors to the nucleus of transduced cells^ref3. While this effect has been demonstrated in both liver and lung, the application of proteasome inhibitors in the context of rAAV2-CFTR gene transfer to the lungs seems to be the closest to practical application. The most likely applications to succeed in the clinical setting may be those in which an existing protein replacement therapy could be supplanted by gene transfer to a nondividing tissue, like skeletal muscle, resulting in sustained secretion of the therapeutic protein into the circulation. Factor IX may be able to prove that concept, but other candidates, such as alpha-1 antitrypsin (AAT) are moving rapidly towards clinical trial. One key to this process is likely to be the avoidance of immune responses to the transgene. If that is the case, proteins like AAT that have not been prone to immune responses in the context of existing protein replacement therapies, are good candidates
• ssRNA negative-strand viruses; Mononegavirales; Paramyxoviridae; Paramyxovirinae; Respirovirus : Sendai virus (SeV) : it uses binding to cholesterol and sialic acid as receptors, which both are present at the apical membrane of airway epithelial cells; in addition SeV is not greatly inhibited by mucus
• ssRNA positive-strand viruses, no DNA stage : Togaviridae : Alphavirus offer several advantageous features, such as a broad host range (including cells of avian, insect, and mammalian origin), high levels of protein expression, and a small genome that is easy to manipulate
• SFV complex
• Semliki forest virus (SFV)
• VEEV complex
• Venezuelan equine encephalitis virus (VEEV)
• WEEV complex
• Sindbis virus
DNA-based alphaviral RNA replicon vectors / suicidal DNA vectors, alleviate the concerns of integration or transformation related to conventional DNA vectors since suicidal DNA vectors eventually cause apoptosis of transfected cells, which may enhance cross-priming of dendritic cells and may also constitute a safety feature that removes the vaccine from tissue. However, the expression of inserted genes in these vectors is transient and lytic, and, despite circumventing the induction of immunological tolerance^ref, the potency of suicidal DNA vaccines may be compromised because of apoptotic cell death : strategies to delay suicidal DNA-induced cell death using antiapoptotic proteins (e.g. SFV encoding antigen fused with BCL-x_L^ref) may greatly enhance the potency of suicidal DNA. However, present strategies for production of such particles have biosafety limitations due to the generation, by RNA recombination, of replication-proficient viruses (RPVs) : a new packaging system for SFV based on a the use of a 2-helper system in which the capsid and spike proteins of the C-p62-6K-E1 polyprotein are expressed from 2 independent RNA molecules has been suggested^ref. The capsid gene contains a translational enhancer and therefore that sequence was also engineered in front of the spike sequence p62-6K-E1. A sequence coding for the FMDV2A autoprotease is inserted in frame between the capsid translational enhancer and the spike genes. This allows production of the spike proteins at high levels with cotranslational removal of the enhancer sequence and normal biosynthesis of the spike complex. The autoprotease activity of the capsid protein was abolished by mutation, further increasing the biosafety of the system. Cotransfection of cells with both helper RNAs and an SFV vector replicon carrying the LacZ gene led to production of recombinant particles with titers of up to 8 x 10⁸ particles per 10⁶ cells. Extensive analysis failed to demonstrate the presence of any RPVs, emphasizing the high biosafety of the system based on 2-helper RNAs.
Please note viral vectors have a limited capacity : so minigenes (i.e. cDNA + promoters) are often used.
Comparison of properties of various vector systems :
 

features	retroviral	lentiviral	adenoviral	AAV	naked/lipid-DNA
maximum insert size	7-7.5 kb	7-7.5 kb	30 kb	3.5-4.0 kb	unlimited size
concentratrions (viral particles per mL)	> 108	> 108	> 1011	> 1012	no limitation
route of gene delivery	ex vivo	ex/in vivo	ex/in vivo	ex/in vivo	ex/in vivo
integration	yes	yes	no	yes/no	very poor
duration of expression in vivo	short	long	short	long	short
stability	good	not tested	good	good	very good
ease of preparation (scale up)	pilot scale up (up to 20-50 L)	not known	easy to scale up (up to 100 Lref)	difficult to purify, difficult to scale up	easy to scale up
immunological problems	few	few	extensive	not known	none
pre-existing immunity	unlikely	unlikely, except maybe AIDS patients	yes	yes	no
safety problems	insertional mutagenesis ?	insertional mutagenesis ?	inflammatory response, toxicity	inflammatory response, toxicity	none

• gene delivery and expression imaging
• aims
• determining the efficiency of given delivery protocols by measuring target organ / background ratio
• establishing viral or non viral vector burden for a given organ
• longitudinal monitoring of vector distribution
• ways
• invasive : requiring a sample of the transfeced tissue, such as a biopsy or brushing sample
• PCR from the transgene DNA
• RT-PCR from the transgene mRNA
• in situ hybridization
• immunohistochemistry (IHC)
• noninvasive : reporter genes : since the first scientist transfected the first plasmid into the first bacterium, molecular biologists have been involved in an ongoing struggle to convince bacteria to retain their episomal baggage.
• genes causing appearance of antibiotic resistance : the most widely used mechanism is the inclusion of an antibiotic resistance gene on the transfected plasmid, and the antibiotic itself in the growth media. Although this works well for small-scale and research applications, it has disadvantages in intensive culture scenarios due to inactivation of the antibody or its dilution. Furthermore, in cases where the final protein or DNA product is to be used for human therapies, the FDA has raised concerns about contamination by antibiotic resistance genes.
• ampicillin resistance (amp^r) is precluded for use in humans
• neomycin resistance (neo^r) : neomycin phosphotransferase (NPT II) => G418 resistance
• kanamycin resistance (kan^r)
• puromycin resistance (puro^R) : N-acetyl transferase (pac)
• tetracycline resistance (tet^r)
• zeocin resistance (zeo^r)
• ccdA/ccdB antidote/poison system : both genes are carried on the transfected plasmid. Since the half-life of CcdA (the antidote) and CcdB (the poison) differ by about 2-fold, the CcdB protein will persist for longer in a cell that has lost the plasmid, eventually killing it. Unfortunately, this is an imperfect system, as division can dilute the effect of the poison, allowing some cells to eventually escape death. To solve this problem and make a more efficient system, the ccbB gene has been moved from the plasmid to the bacterial genome by creating a ccdB⁺ strain of DH10B. In the presence of a transfected plasmid carrying only the ccdA gene, repression of ccdB poison gene expression occurs, while in its absence, expression of ccdB is up-regulated, leading to cell death. This separate-component-stabilization (SCS) strategy: (i) allows 100% stability of transfected plasmids without the use of antibiotics; (ii) increases 3-5 times the recombinant protein production levels; and (iii) does not require any specific modification of the protein production process or culture medium. We illustrate that point by using the classical T7 promotor (i.e., used in most expression systems). Finally, the SCS system increases by 5 the yield in DNA production, a result especially important for the design and production of gene therapy constructs void of any antibiotic resistance gene^ref.
• genes coding for fluorescent proteins. Advances in imaging using optical imaging devices have made possible the use of optical techniques to analyze, in real time, reporter gene expression in small animals such as mice. Although imaging reporter gene expression by optical tehniques has attenuation, scatter, and resolution limitations, it is convenient, fast and relatively inexpensive. However, the potential use of in vivo optical imaging of visible proteins is substantially limited in humans, due to attenuation of visible light : anyway it can be detected with fluorescence video endoscope or laser-induced fluorescence endoscope in combination with a CCD camera.
• genes coding for non-endogenous enzymes which transform non-endogenous substrates into products that are ...
• directly visible
• D-luciferin ==firefly (Photinus pyralis) luciferase==>
• 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) ==Escherichia coli b-galactosidase / lacZ==> blue precipitate
• detectable by spectrophotometry
• PNPP + H₂O =[human placental secreted alkaline phosphatase (SEAP)]_serum=> PNP + P_i
• radioactive labeling with a g-emitting isotope => detection by a g-camera
• heterobifunctional peptide-based DNA binding chelates (PBC)
• Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Lys₄-Lys-(N-e-[4-(psoralen-8-yloxy)] butyrate)-NH₂ labeled with a g-emitting isotope
• MRI-based strategies
• PET-based strategies : the location, magnitude and duration of the PET reporter gene product (enzyme, receptor, ...) expression is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled ligand (substrate, messenger, ...) of the reporter gene product (PET reporter probe). E.g.
• PET reporter gene : HSV-1tk
• PET reporter probe :
• thymidine analogues
• 2'-[¹⁸F] fluoro-2'-deoxy-1-b-D-arabinofuranosyl-5-iodouracil (FIAU)
• acycloguanosine antiviral agents
• 8-[¹⁸F] fluoroganciclovir
• 8-[¹⁸F] fluoroacyclovir
• 8-[¹⁸F] fluoropenciclovir
• 9-[(4-[¹⁸F]fluoro-3-hydroxymethylbutyl)guanine
• physiological strategies : change in levels of endogenous metabolites
• [bilirubine]_plasma : bilirubin-uridinediphosphoglucuronate (UGT1A1) in jaundiced Gunn rats (UGT1A1^-/-, a model of human Crigler-Najar syndrome type 1)
• [tyrosine]_plasma : fumarylacetocetate hydrolase (FAH) in FAH-deficient mice (a model of human hereditary tyrosinemia type I)
• pigmentation change in melanocytes (Melan-C) derived from the albino mouse, which contain an homozygous point mutation (TGT => TCT) in the tyrosinase gene.
• measuring the correction in transepithelial voltage in CF patients by nasal voltage measurements or in large airways.
• clinical, radiological and endoscopic regression in tumor volume
• multifunction reporter genes :
• bifunctional
• HSV-1tk and North American firefly (Photinus pyralis) luciferase (ffLuc)^{ref1, ref2}
• fusion gene of hygromycin phosphotransferase and HSV-1 thymidine kinase (HyTk)^ref
• trifunctional ffLucHyTK gene
• post-transfection control of gene expression
• promoters
• strong constitutive viral promoters (high basal transcription rates; neither tissue nor time specificity ; LTRs tend to be silenced by IFN-g and TNF-a produced during immune responses)
• HHV-5 / human cytomegalovirus (CMV) (HCMV) immediate-early (IE) (CMV-IE) enhancer-promoter (known as the CMV promoter) (HCMV IEp : inactivated in MoMV vectors but not in Lentivirus vectors)
• CMV/IA (intron A)
• Moloney murine leukaemia virus (MoMLV / MoMV / MMLV / MuLV / MLV) 5' LTR promoter
• RSV 3' LTR promoter
• RSV U3 enhancer
• RSV-TAR (transactivation response element)
• SV40 promoter (weaker than CMV-IE)
• CaMV promoter
• Syn1 promoter
• HIV-1-LTR
• murine leukemia virus (SL3-3) promoter
• SV2
• AKV murine leukemia viral LTR
• combinations of the abovementioned promoters/enhancers
• hybrid promoters
• CMV + ubiquitin B (UbB)
• hybrid b-actin promoter/CMV enhancer
• mammalian promoters
• strong aspecific house-keeping promoters
• hEF-1a₁ promoter
• ubiquitin C (UbC) promoter
• phosphoglycerate kinase-1 (PGK-1) promoter
• desmin
• condition-specific promoters
• hypertonicity-sensitive promoters
• NFAT5 (?)
• temperature-sensitive promoters
• pL promoter from l bacteriophage
• ionizing-radiation (IR)-responsive elements
• CDKN1A / p21 / WAF1 promoter
• promoters containing the CCTTATTTGG motifs (CArG elements / serum responsive elements (SREs)), which can be arranged as multiple copies in synthetic promoters. Best results are obtained by using 9 tandem-repeat copies of the altered CCATATAAGG sequence (E9-ns2). Cellular stresses such as radiation cause production of reactive oxygen species and oxidative chemicals (such as H₂O₂), able to activate the MAPK cascade, which in turn phosphorylate the serum response factor (SRF) : then it can bind to SREs, complexing with p62/TCF, SAP-1 and Elk-1.The expression of SRF gene itself is also partially self-regulated via CArG elements situated in its own promoter. However, maximal serum responsiveness requires the presence of the Sp1 binding site immediately adjacent to one of the 3 CArG elements.
• Egr1 / zif268 : itcontains several potential Sp1 binding sites, one of which is located between 2 IR-responsive CArG elements.
• c-fos
• hypoxia-responsive element (HRE) containing the core motif 5'-(A/G)CGT(G/C)(G/C)-3'. Typically they contain several transcription factor binding sites, one of which is essential for hypoxic activation by binding the complex hypoxia-inducible factor 1 (HIF-1). HIF-1 functions as a heterodimer consisting of 2 bHLH proteins, HIF-1a and HIF-1b / aryl receptor nuclear translocator (ARNT). Although both subunits are constitutively expressed, HIF-1a is hypoxia-regulated via the posttranslational stabilization and transactivation by several additional factors. The HRE/HIF-1 regulation system is common to all mammalian cells and human tissues tested and the HIF-1a subunit overexpressed in 68-84% of the tumor types analyzed. Hypoxia responsive promoters come from ...
• phosphoglycerate kinase-1 (PGK-1) : best results with 5 HREs
• lactate dehydrogenase A (LDH-A)
• VEGF-A : best results with 5 HREs
• Epo : best results with 9 HREs
• both hypoxic and radiation response elements
• glucose transporter-1 (GLUT-1)
• tissue-specific promoters / tissue-specific elements (TSE) (transcriptional targeting) : gene is not switched on in tissues other than the intended one. However, tissue-specific promoters are not absolutely specific and within a complex biological system there may be a certain amount of "promoter leak".
• lens
• d2-crystallin
• chicken b-actin promoter
• neurons
• NSE
• astrocytes
• GFAP
• T lymphocytes
• CD2
• Lck
• melanocytes
• tyrosinase
• endothelial cells
• vWF
• Flt-1
• skeletal muscle cells (SMC)
• myosin light chain (MLC) 3F promoter
• muscle-specific creatine kinase (CKM) promoter
• smooth muscle cells
• SM22a
• cardiomyocytes
• a-myosin heavy chain (aMyHC)
• both slow-twitch skeletal muscle and some nonmuscle cell types
• ventricular/slow-twitch skeletal muscle myosin light chain 1
• hepatocytes
• albumin
• transthyretin
• adipocytes :
• FABP4
• pancreatic b-cells
• insulin (e.g. rat insulin promoter (RIP))
• exocrine pancreas epithelial cells
• elastase 1, pancreatic
• keratinocytes
• involucrin (INV)
• keratinocytes and thymic epithelial cells (TECs)
• keratin 14 (K14)
• mature dendritic cells
• fascin^{ref1, ref2}
• prostatic epithelial cells
• a combination of a rat probasin promoter + a human KLK3 / PSA promoter
• PSP94 / b-microseminoprotein
• prostate alkaline phosphatase (PAP)
• rat PSBP3 / C3(1)
• locus control regions (LCR) are tissue-specific regulatory elements which are able to overcome chromatin position effects and confer upon a gene linked in cis, site-of-integration, physiological levels of expression directly proportional to transgene copy number in mice. LCRs are able to obstruct the spread of heterochromatin and prevent position effect variegation (PEV). This pattern of expression conferred by LCRs suggests that these elements possess a powerful chromatin remodelling capability and are able to establish and maintain a transcriptionally competent, open chromatin domain. Therefore, LCRs are seen as the most efficient means of achieving reproducible, physiological levels of gene expression from stably integrated transgenes.
• b-globin LCR
• IgH LCR
• dividing endothelial cells-specific promoters (angioblasts, physiological and neoplastic angioneogenesis)
• Flt-1 / VEGFR1 promoter
• Flk-1 / VEGFR2 / KDR promoter
• endoglin promoter
• tumor-specific promoters are those of genes coding for tumor antigens. The low strength of a tumor-specific promoter can be bypassed by using binary promoter systems : a tumor-specific promoter can drive expression of a chimeric transcription factor consisting of the powerful herpes simplex virus VP16 transcriptional activation domain fused to the DNA-binding domain of the yeast protein GAL4, which then binds to the GAL4-binding sites upstream of a minimal promoter to activate transgene expression.
• time-specific transcriptional complexes
• glucose-activated
• isopropylthio-b-D-galactoside (IPTG)-activated
• Lac operon promoter
• temperature-sensitive (ts) alleles engineered using ts splicing variants of Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred into Gal80. Gal80-inteinTS is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster. Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.
• gaseous acetaldehyde-inducible regulation (AIR) : AlcR-P_alcA transactivator-promoter interaction of the Aspergillus nidulans ethanol-catabolizing regulon^ref
• drug-activated
• G_sPCR ligands, CREB and cAMP response elements (CRE) in the transgene promoter
• Tet-on : tetracyclines (doxycyclin), transactivator rtTA and tetracycline responsive element (TRE) in the minimal CMV promoter of the transgene. Basal activity can be reduced by :
• substituting the minimal CMV promoter with a modified MMTV promoter
• flanking the inducible promoter with insulator elements
• co-transfecting tTS trascriptional silencer (a fusion protein of the tet repressor protein (TetR) and the KRAB-AB domain of the Kid-1 protein) that binds to the tetO sequence in the TRE region in absence of doxycyclin and minimize basal expression
• [(DNA-binding domain ZFHD1 + immunophilin FKBP domain) --- (FRAP rapamycin-binding domain + p65 RelA transcriptional activation domain)] and rapamycin.
• CuSO₄-activated
• drug-inhibited
• Tet-off : tetracyclines (doxycyclin)
• by combining 2 repressors, which control each other's expression, an epigenetic circuitry able to switch between 2 stable transgene expression states after transient administration of 2 alternate drugs is created. Engineered Chinese hamster ovary cells (CHO-K1) showed toggle switch–specific expression profiles of a human glycoprotein in culture, as well as after microencapsulation and implantation into mice. Switch dynamics and expression stability could be predicted with mathematical models^ref
• artificial zinc-finger protein transcription factors (TFs_ZF or ZFP TFs). Individual zinc fingers are short stretches of 30 amino acids folded into compact structures. Most natural ZFP TFs have 3 fingers, but some have as many as 37, arranged one after another, like fingers on a hand, positioned in such a way that they can readily touch multiple, adjacent spots along the DNA double helix. The key to zinc fingers' success is their sequence-specific, modular structure : with each zinc finger targeting a specific 3-bp DNA sequence, a 6-fingered protein, for example, should have the capacity to bind just one of nearly 70 billion unique 18-bp sequences. That's more than good enough to target a single gene in the 3.2 billion-bp human genome. Multizinc finger peptides are likely to reach increased prominence in the search for the "ideal" designer transcription factor for in vivo applications such as gene therapy. However, for these treatments to be effective and safe, the peptides must bind with high affinity and, more importantly, with great specificity. Zinc finger arrays can be made to bind 18 bp of DNA with pM affinity, but also has suggested that arrays of fingers also may bind tightly to related sequences. By linking 3 2-finger domains rather than 2 3-finger units far greater target specificity can be achieved through increased discrimination against mutated or closely related sequences. These new peptides have the added capability of being able to span 2 short gaps of unbound DNA, although still binding with picomolar affinity to their target sites. Other ways to engineer transcription factors exist, but none of them match the combinatorial power of ZFP TFs. In addition to their combinatorial power, by mixing and matching regulatory domains with zinc fingers, scientists can engineer ZFP TFs that either activate or inhibit gene expression. When used to overexpress endogenous genes, ZFP TFs produce all the splice variants in their proper ratios : the resulting phenotype is much more natural than that attained by transgenic expression, which produces only a single splice variant.
• transcriptional silencing, one of the major impediments to gene therapy in humans, is often accompanied by replication during late S-phase. Transcriptional silencing and late replication were prevented by DNA sequences that can initiate DNA replication (replicators). When replicators were included in silencing-prone transgenes, they did not undergo transcriptional silencing, replicated early and maintained histone acetylation patterns characteristic of euchromatin. A mutant replicator, which could not initiate replication, could not prevent gene silencing and replicated late when included in identical transgenes and inserted at identical locations. These observations suggest that replicators introduce epigenetic chromatin changes that facilitate initiation of DNA replication and affect gene silencing. Inclusion of functional replicators in gene therapy vectors may provide a tool for stabilizing gene expression patterns^ref
Comparison of promoters used in DNA expression studies in vitro and in vivo :

expressed antigen	promoters/enhancers compared	in vitro/in vivo comparison
GFP	CMV, muscle-specific creatine kinase (CKM) promoter	Consistently higher levels of GFP expression were driven by the CKM promoter compared to CMV in miceref
LacZ	CMV, glial fibrillary acidic protein (GFAP) promoter, neuron-specific enolase (NSE) promoter	Injection of mice with the constructs containing the different promoters showed that GFAP is as efficient at driving lacZ expression as CMVref.
CAT	HIV-1-LTR (long terminal repeat), RSV-TAR (transactivation response element)	HIV-1-LTR could be transactivated by tat in both stimulated and unstimulated cells; RSV-TAR was only transactivated in unstimulated cellsref.
CAT	CMV, RSV, SV40, murine leukemia virus (SL3-3) promoter	The CMV promoter was found to be stronger than any of the other promoters tested in muscleref.
CAT	CMV, SV2	The CMV promoter was found to have greatest transcriptional activityref.
Luciferase	CMV, RSV, SV40, PGK, hybrid b-actin promoter/CMV enhancer, CMV/IA (intron A)	The hybrid b-actin/CMV promoter/enhancer showed greater luciferase expression than RSV, SV40, PGK or CMV. CMV/IA also showed 2–6 fold in vitro and 1.5–3 fold in vivo higher luciferase expression than CMVref.
Hepatitis B surface antigen  (HBsAg)	CMV, desmin	The promoters performed equally well in vitro, and CTL and Th1 serum antibody responses against HbsAg in mice were of similar magnituderef.
Hepatitis B envelope proteins	CMV, desmin	Greater in vitro expression of antigen was attributed to the desmin promoter. However, comparable humoral and cytotoxic immune responses were stimulated following i.m. injection of miceref.
Rabies virus G protein	CMV, SV40	Comparable G antigen-specific antibody titres were stimulated in mice. Slightly higher T cell responses were observed from the CMV constructref.
Influenza virus H5 hemagglutinin  (HA)	CMV, b-actin	Constructs containing the CMV or b-actin promoters provided comparable protection against influenza in chickensref.
Influenza virus H5 hemagglutinin  (HA)	CMV, b-actin, RSV, SV40	Similar in vitro expression of HA. The greatest HA-specific antibody and protection against influenza in chickens was provided with the CMV construct.
Bovine herpesvirus glycoprotein  D (gD)	RSV, CMV/IA	CMV/IA construct produced higher neutralising antibody titres against gD in i.d. injected cattleref.
HIV-1 gag/env	CMV, AKV murine leukemia viral long terminal repeat	CMV showed 10–20 fold greater activity than AKV in vitro. Immunised macaques developed high humoral responses with the CMV construct onlyref.
SV40 large tumour antigen	CMV, SV40	The CMV construct induced higher levels of antibody and protection in the murine experimental metastasis model than the SV40 constructref.
M. tuberculosis apa + pro  proteins	CMV, UbC	The CMV promoter was the most efficient testedref.
Adenovirus E4 ORF3	CMV, RSV, SV40, UbC, EF-1a	Following i.n. dosing to mice, constructs containing the UbC and EF-1a promoters stimulated the most stable expression of antigenref

• post-transcriptional control of gene expression
• polyadenylation signals
• bovine growth hormone (BGH) 1 polyA site
• SV-40 late polyA site
• riboregulators : complementary cis sequence inserted directly upstream of the ribosome binding site in a target gene. Upon transcription, this cis-repressive sequence causes a stem-loop structure to form at the 5'–UTR of the mRNA which interferes with ribosome binding, silencing gene expression. A small noncoding RNA that is expressed in trans targets the cis-repressed RNA with high specificity, causing an alteration in the stem-loop structure that activates expression. Such engineered riboregulators may lend insight into mechanistic actions of endogenous RNA-based processes and could serve as scalable components of biological networks, able to function with any promoter or gene to directly control gene expression^ref.
Strategies to coordinately co-express 2 or more transgenes (e.g. required when a reporter/marker gene is needed in order to track the expression of the therapeutic gene (normally not easy to detect), or in the correction of multi-gene diseases that involve heteromultimeric proteins, several enzymes involved in a biochemical pathway or various proteins for combined/synergistic effects) :
• separate promoters can be used to drive expression of different genes in the same vector to achieve coordinated maintenance of gene copy number within cells. However, such constructs often suffer from the problem of promoter attenuation, whereby the 3' gene is suppressed upon selection of the 5' gene and vice versa.
• splicing
• in-frame fusion of 2 different genes to produce a chimeric protein, guaranteeing the expression of both genes simultaneously. It is the polyprotein strategy that many viruses use to co-express most of their proteins, or even all of them (as in picornaviruses). Not surprisingly, this strategy is indeed used by cells, although not very often, in particular for the co-ordinated secretion of different proteins and peptides. Recently, several groups have been trying to utilize this co-expression strategy. One of the possibilities is to introduce the target site for a cellular proteinase between 2 cistrons cloned in frame forming a single open reading frame (ORF)^ref. In this way the polyprotein is synthesized as a fusion protein that post-translationally is proteolytically cleaved to yield the discrete proteins of interest. Unfortunately, this strategy has several practical difficulties:
• the polyprotein to be cleaved must reside, or at least pass through, the same compartment as the proteinase
• the cleavage is not always independent of the context
• the cleavage may be incomplete and unpredictable
• efficient cleavage will only be produced in cells actively expressing the proteinase
• the post-translational cleavage is not compatible with all possible sub-cellular targetings.
Although some functional fusion proteins have been succesfully expressed, eg the ADA-MDR1 fusion protein, this strategy may not work for all combinations of proteins, some of which could result in protein misfolding or mistargeting.
• reinitiation : a co-translational strategy independent of cellular or viral factors. Ribosomes first translate an upstream cistron, although highly inefficiently, then resume translation of the second, downstream, cistron. Reinitiation was indeed tried in the 1980s, but the large imbalance makes it unsuitable for co-expression of even 2 genes^ref. These first methods were all reported in 1980s^ref. During the 1990s, nearly all those strategies were abandoned in favour of :
• bicistronic constructs, in which 2 genes separated by an internal ribosome entry site (IRES) sequence are expressed as a single transcriptional cassette (or bicistronic transcript) under the control of a common upstream promoter. IRES sequences have been found naturally in the 5' UTR of :
• viral genes from Picornaviridae :
• encephalomyocarditis virus (EMCV)
• polioviruses
The recognition of their secondary RNA structure allows them to be translated even after disruption of normal mRNA capping mechanism at the E4 subunit. IRESs are ~ 450 nt in length.
• mammalian genes : IRESs can range in size from ~ 100 bp to > 1 Kb. Unlike viral IRESs, mammalian IRESs often serve as recognition sites for transcriptional regulatory factors, thereby mediating post-transcriptional regulation of gene expression.
• BiP / hsp70 5 / MIF2 / GRP78 : like the ECMV IRES, it contains a Y-type stem-loop structure. Unlike the ECMV IRES, however, the BiP IRES has an additional hairpin immediately before the authentic AUG start codon and does not have a polypyrimidine tract (PPT): although the SSB / La autoantigen and the PPT binding protein (PTB) 1 and 2 have been reported to be important for efficient EMCV IRES function, these 2 proteins do not seem to interact with  BiP IRES.
• eIF4G 1, 2 and 3: 100-fold better than viral IRESs in mediating downstream gene expression in most cell lines. It is structurally similar to ECMV IRES, and contains cryptic AUG start sites and a functional PPT that is required for activity. However, unlike the EMCV IRES; the PPT of the eIF4G IRES cannot be replaced by purines. Another feature that distinguishes the eIF4G IRES from the ECMV IRES is the ability of eIF4G to inititate translation when the AUG start is variably spaced from the PPT, whereas the spacing between the PPT and the initiator AUG in the EMCV IRES is critical for downstream gene expression. These features of the eIF4G IRES could perhaps account for its superior efficiency in mediating downstream gene expression.
• c-Myc : more complex secondary RNA structure and absence of cryptic translation start sites
• VEGF-A, whose translation remains efficient even under unfavorable hypoxic and/or hypoglicemic conditions when overall translation is reduced. The VEGF 5' UTR is very unique in that is contains 2 independent IRESs, A and B, acting together to facilitate efficient initiation of translation at the same AUG codon. IRES A is approximately 300 bp in length and located immediately upstream from the initator AUG, thus allowing for ribosome binding and translation initiation without scanning. IRES B is located upstream of IRES A and is approximately 400 bp in length. It contains a predicted stem-loop structure bearing the GNRA motif characteristic of picornavirus IRESs and a PPT. Different proteins bind to each of the 2 IRESs, with very few proteins binding to IRES A, the majority of proteins including PTB binding to IRES B. However, whereas PTB is important for the function of the ECMV IRES, its binding to IRES B of VEGF is not correlated with IRES activity.
• FGF-2 / bFGF, whose translation is stress-modulated.
Such a design enables coupled transcription of both genes, followed by cap-dependent inititation of translation of the first gene and IRES-directed cap-independent translation of the downstream genes from the same mRNA. In bicistronic mRNAs bearing an IRES sequence, the first cistron is translated by scanning ribosomes that enter via the 5' end. The cloning of an IRES sequence downstream of the first cistron, allows the internal entry of ribosomes that translate the second cistron. As each cistron is translated from a different translational initiation event, both translations are uncoupled, and the proteins are not obtained in an equimolecular proportion ("imbalance") leading to a large excess of the first protein. The drive to co-express more than 2 genes, opening the door to therapies for muti-gene deficiencies, was halted by the inability of vector technology to guarantee a reliable co-expression. Nevertheless, IRESs were the first strategy that met with some success, and several polycistronic vectors able to co-express up to 4 genes were developed during the 1990's^ref. However, 2 main problems blocked the successful use of large and complex polycistronic vectors: the large size and imbalance of most IRESs which makes it very difficult to predict the level of expression of the downstream cistron^ref.
• cis-acting hydrolase element (CHYSEL) : Picornaviridae, the same family of viruses to first provide the IRESs, encode all their proteins in a long single ORF that is cleaved post-translationally by viral proteinases. However, it was described in the 1980's that at one position, the polyprotein of some picornaviruses (such as FMDV) underwent a rapid co-translational self-processing. It was soon realised that the key was a small 18aa peptide (2A) that directed its own separation from the growing polyprotein. During the last decade, this mechanism has been studied in detail, resulting in a simple model: the small 2A peptide, during its translation, interacts with the exit tunnel of the ribosome to induce the "skipping" of the last peptide bond at the C-terminus of 2A. The crucial point is that the ribosome is able to continue translating the downstream gene, after releasing the first protein fused in its C-terminus to 2A. From a biotechnological standpoint, all that is needed is to clone the coding sequence of 2A, followed by the codon for the first amino acid of the next FMDV protein (2B), in frame between the 2 genes one wishes to co-express. The synthesis of the peptide bond between the last amino acid (Gly) of 2A and the first (Pro) of 2B is skipped, producing an upstream protein with a C-terminal tail of 18aa (2A) and a downstream protein with a Pro at the N-terminus. The extra sequences have minimal effect on the activity of most proteins and none on their stability. In fact, the 2A peptide has been used as an efficient tag for immunoprecipitation and Western blotting, although commercial antibodies are not yet available. Interestingly, additional CHYSEL sequences have been found in viruses other than FMDV. Broad applicability of 2A. The initial publications using this strategy have shown that 2A skipping can be used in the typical viral vectors used for gene therapy (retrovirus and adeno-associated virus) to reliably co-express many reporter proteins (NEO; puromycin N-acetyl transferase, PAC; GFP, etc) and therapeutic proteins (HSV1TK; IL-12; viral antigens, etc.) in transient transduced or stable cells lines and in animals. A full list of publications using 2A is available on the web^ref. Several publications in the past few months have shown the potential of this new co-expression strategy^{ref1, ref2, ref3}. Co-ordinating the co-expression of all your genes. Up to 4 genes have been successfully co-expressed from plasmids and retrovirus using several copies of the FMDV 2A or other 2A-like sequences (to avoid direct repeats in retroviruses)^{ref1, ref2}. Not only was co-expression effective, its co-ordination was also apparent^{ref1, ref2}, and the imbalance in the level of the proteins expressed was low (determined to 1.2^ref).


These properties allowed polycistronic vectors bearing pac in the last position to easily generate stable cell lines co-expressing two upstream genes^{ref1, ref2}. Putting your proteins where they should be. The CHYSEL strategy of co-expression is also compatible with the most disparate sub-cellular localisations^{ref1, ref2, ref3, ref4}. Proteins processed by 2A from polyproteins were targeted to the cytosol, nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, plasma membrane (both, by transmembrane proteins and by cytosolic attachment due to myristoylation) and the extra-cellular compartment. Post-translationally targeted cytosolic proteins as well as co-translationally secreted and transmembrane proteins type I, II and III, have been successfully co-translated. Only one combination of co-translational signals was not correctly targeted^ref. Designing complex polyproteins for multi-gene deficiency. The results reported^ref should be particularly interesting for researchers in the gene therapy field. They provide a good example of the potential of the 2A co-expression strategy, introducing up to 4 genes in a single vector. Furthermore, they show the utility of this strategy to reconstruct a very delicate heteromultimeric protein complex on the cell surface, T-cell receptor:CD3 complex (TCR:CD3). It is known that all 6 subunits are necessary for the efficient formation of the TCR:CD3 complex and just 2 retroviral vectors were sufficient to reconstruct it in transfected 293T or infected 3T3 cells: one encoding both subunits of the TCR and the other the 4 subunits of the CD3 complex.

Lethally irradiated CD3e^DP/DP × CD3z^-/- mice (lacking all 4 CD3 subunits) were transplanted with bone marrow from wt C57BL/6 mice or CD3e^DP/DP × CD3z^-/- mice transduced with a retrovirus encoding the four CD3 subunits, and in both cases TCR surface expression was detected and the T cells proliferated normally after immune stimulation. Bone marrow from CD3e^DP/DP × CD3z^-/- mice without CD3 transduction did not restore T-cell development. T cells were also reconstituted in sub-lethally irradiated RAG-1^-/- mice (lacking mature T and B lymphocytes) in which bone marrow from CD3e^DP/DP mice (lacking CDe and with a severe inhibition of CD3g and CD3z), transduced with a retrovirus encoding these 3 subunits (via 2 2A sequences), was used for a transplant into the RAG-1^-/- mice. The same experiment using three vectors encoding the CD3 subunits separately was unsuccessful. The development of FMDV 2A as a cloning tool is an example of how dangerous pathogenic viruses can be harnessed by biotechnology for human benefit. Their molecular "tricks" (as IRES or CHYSEL sequences) are gradually becoming part of the biotechnologists' toolbox. The development of the polycistronic vectors here discussed is a big step forward, a decade and a half after the launching of the very first gene therapy trial with the aim of introducing in blood cells just a single therapeutic gene, adenosine deaminase (ADA), and the NEO marker^ref.
• Strategies for controlled enzyme activation : transfected gene codes for a fusion protein composed of an intracellular signaling domain and a ligand binding domain (LBD). The ligand can be:
• monovalent
• bivalent : such ligand rapidly crosslinks 2 chimeric signaling molecules that are fused to LBD. The ligand can be ...
• hormone (hormone binding domain (HBD))
• synthetic ligand (chemically induced dimerization (CID))
The 2 chimeric signaling molecules can be ...
• identical (homodimerization). E.g. :
• Mpl signaling domain + FKBP, switched on by homodimerization induced by ...
• G-CSFR signaling domain and ER hormone-binding domain, switched on by estrogen- or tamoxifen-induced homodimerization (GCRER)
• different (heterodimerization). E.g. :
• inducible Akt / PKB (iAkt) :
• LBD : rapamycin-binding domain from ...
• FKBP12
• S6 kinase kinase FRAP / mTOR_2025-2113 (FRB₁, containing modified T2098L).
It binds to ...
• FK506
• FK1012 (potential interactions with endogenous FKBP12 can lead to immunosuppression of recipients !)
• rapamycin
• rapalogs
• AP1903
• AP20187
• AP22783
While rapalogs bind with high affinity to FRB₁, binding to endogenous FRAP / mTOR is sterically prevented, so they are bioinert.
• rest of chimera 1 : Akt / PKB signaling domain (without PH domain) [DPH.Akt]

rest of chimera 2 : c-Src myristoylation signal (to effect membrane recruitment, replacing PH domain function) [M-FRB₁ or M-(FRB₁)₂]

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