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