Clinical gene transfer had its official beginning when, in 1989, 5 patients with terminal melanoma were given autologous lymphocytes that had been "marked" ex vivo with a gene encoding resistance to the antibiotic G418. This study was designed primarily to trace the cells in the patients' bodies and to show the safety of gene transfer, and in that sense it was successful. No helper viruses were found, no reverse transcriptase activity was detected, no toxicity was experienced, and the transduced cells remained otherwise "normal"^ref.

Principles of gene therapy

Gene therapy consists of altering cell genotype by transfecting polynucleotides ...
• ... directly into body cells (in vivo gene therapy)
• ... into cells extracted, cultured in vitro, transfected, selected (also against deleterious insertional mutagenesis !) and then re-inserted into the donor's body (ex vivo gene therapy, sometimes wrongly called in vitro gene therapy) : please note that not all cell types can be extracted from the body (e.g. neurons) and/or cultured in vitro (e.g. pneumocytes) and/or survive for a long time in the body (e.g. RBCs) !
Vectors :
• gene transfer agents (GTAs)
• gene delivery and expression imaging
• expression control
Vector administration routes : (re)insertion can be preceeded by partial ablation (e.g. in bone marrow) to allow space creation. (Re)insertion can be done by ...
• portal vein infusion => liver
• stereotaxic injection in brain
• catheterism => targets the endothelium : the DNA that has not been uptaken is eliminated via the urinary tract
• endotracheal microspraying => aerosolization => targets the epithelium of the lungs : the DNA that has not been uptaken is elminated via the feces
• nasal instillation
• lobar instillation
Recipient cells may be ...
• from an ontogenetic point of view :
• ... somatic cells => somatic gene therapy
• prenatal or in utero somatic gene therapy is technically possible with reasonable safety, depending on the target site and time of application.
• embryo somatic gene therapy
• fetal somatic gene therapy
Routes for mammalian embryos :
• ultrasound-guided injection in ...
• embryo / fetus => transfection is found only in the regions peripheral to the site of injection
• placenta
• amniotic cavity => transfection is found only in surface tissues
• yolk sac vessels
• umbilical cord
• circulation of pregnant mother => transfects a variety of embryonic tissues but also undesirable gene delivery to the mother
• tail vein injection in mice => flow of DNA from maternal circulation to fetus ...
• ... before establishment of placental circulation (egg cylinder stage, before E9-10) : transferred to visceral endoderm or yolk sac by endocytosis or pinocytosis. The majority of these conjugates may be trapped in the visceral endoderm, but some may be transferred to the fetus via the vitelline circulation or by diffusion
• ... after establishment of placental circulation (E11.5 and thereafter) :
• flow beyond blood-placenta barrier => umbilical vein (within the umbilical cord) => embryo
• flow from deciduum to yolk sac
Advantages :
• avoidance of early disease manifestation
• allow targeting of otherwise inaccessible organs or tissues with relatively low vector doses
• achieve permanent gene expression by use of integrating vectors in still expanding stem cell populations : however even high level transient expression may e.g. safeguard against the hazards of birth trauma in hemophilia.
• prenatal tolerance may avoid immune reactions against vector and transgenic protein also in postnatal life
• postnatal somatic gene therapy
• ... germinal cells => germinal gene therapy. Nowadays it is forbidden in every country. There are only 2 conditions in which the probability of getting an affected offspring is very near to 100% :
• homoplasmy for a dominant mtDNA mutation in the mother. Here gene therapy appears uneffective because of the presence of many mitochondria in each egg !
• union between individuals homozigous for a recessively inherited disease. Here in vitro fertilization and selection of an unaffected embryo appears a more rational solution than gene therapy on affected embryos !
When practicing somatic gene therapy the risk of inadvertent germ-line gene transfer is minimal.
• from a functional point of view :
• ... the cell to be targeted itself
• ... a cell near the cell(s) to be targeted, that partecipates in killing of the targeted cell(s) (e.g. fibroblasts)
• ... a long-life distant cell that acts as a reservoir for the gene encoding a protein secreted into the bloodstream (secretion gene therapy (SGT)). The easy-to-reach cells are
• epithelial cells lining airways, which can be transfected in vivo by inhaling sprays and myocytes (as muscle tissue has large mass).
• epithelial cells lining GI tract, which can be transfected in vivo by swallowing pills (see Genteric, Inc. technology)
• ... a cell to which a selective advantage is conferred to survive killing of all other cells
• from a therapy-lasting point of view :
• ... a cell undergoing turn-over : therapy is effective only if it involves stem cells or if the aim is a rapid cell killing (e.g. for tumor cells)
• ... a permanent cell (e.g. neuron, myoblast) eventually encapsulated into hollow microporous polymer membranes : therapy is definitive if gene expression is long-lasting.
The aim of the gene therapy is to reverse pathogenesis by ..
• classical gene therapy
• increasing gene dose in deficient cells : it is expecially effective for
• oncosuppressor genes :
• TP53 (INGN 201 / Ad-p53 / Ad5CMV-p53 / Adenoviral p53 / INGN 101 / RPR/INGN 201^ref (Advexin^®; source : Introgen's ) has 18 patents) : the first randomised phase II/III trial testing this approach in patients with ovarian cancer was closed early because the treatment did not provide adequate therapeutic benefit : dominant negative "cross talk" between p53 mutants that are overexpressed in ovarian cancer, coupled with aberrant silencing of genes, may have compromised the effectiveness of p53 gene therapy^ref. On the other side a advanced non-small lung cancer was treated by a local injection of ADVEXIN once every 4 weeks for 14 times without marked adverse events, which resulted in tumor regression and relief of his symptom for a year^ref
• a recombinant adenovirus carrying human wild-type p53, GM-CSF, and B7-1 genes (Ad-p53/GM-CSF/B7-1)
• suppressive effect on the growth of laryngeal cancer cells
• inhibits growth of multiple myeloma cells via enhanced apoptosis and the immunogenicity of tumor cells was augmented. The combinatorial effect of these 3 genes on inducing cytotoxic T lymphocytes (CTLs) was more evident than that of p53 individually or any combinations of 2 (p53 + GM-CSF or p53 + B7-1). Furthermore, significant proliferation of autologous peripheral blood lymphocytes (PBLs) and specific cytotoxicity against autologous primary MM cells were induced in vitro^ref
• diseases with recessive inheritance (low levels of gene expression are required, whereas in diseases with dominant heredity very high levels of gene expression are required, ie > 50% of the wild-type homozygote expression level !). E.g. Tyr hydroxylase in Parkinson's disease (PD)
• induction of immune tolerance : CTLA4Ig fusion gene delivery and expression in vivo using retrovirally transduced (using the centrifugal enhancement method) myeloid dendritic cells that induce alloantigen-specific T cell anergy in vitro by blocking expression of CD80 and CD86, costimulatory signals necessary for naive T cell activation^ref
• killing of degenerated cells : e.g. cancer gene therapy, virus-infected cells, T cells mediating GvHD after BMT...).
• direct killing by suicide gene therapy (a particular kind of genetic ablation), by transfering genes coding for ...
• ... NOS2 / iNOS : NO^. is an hypoxic cell radiosensitizer. Spontaneous and bioreductive NO^. donors can't administered in sufficient doses to achieve tumor radiosensitization in the clinic as they elicit systemic vasodilation and hypotension.
• ... a cytotoxin (toxigenic approach)
• ... a proapoptotic gene (Bax, TRAIL : alternative application of genes involved in different apoptotic pathways or different cell killing models may help to overcome development of resistance)
• ... a channel for entry of radioisotope(s) : e.g. Na⁺/I^- symporter (NIS) to increase radioactive iodide (¹³¹I) uptake (RAIU). In fact it is generally believed that the therapeutic effectiveness of ¹³¹I for patients with thyroid cancer largely depends on the unique ability of thyroid tissues to organify ¹³¹I (increaseable by co-delivery of thyroid peroxidase (TPO)). Prolonged ¹³¹I retention is thought to be essential to achieve sufficient therapeutic dose. However, therapeutic dose delivered to tumors could also be increased by increased ¹³¹I uptake and the use of alternative radionuclides with more potent radioactivity (e.g. ¹⁸⁸ReO₄). It has been proved for medullary thyroid carcinoma^ref
• ... a prodrug activator (chemogenes) that converts in situ a relatively harmless prodrug into a cytotoxic drug (gene-directed enzyme prodrug therapy (GDEPT) or genetic prodrug activation therapy (GPAT); when viral vectors are used it is a.k.a. virus-directed enzyme prodrug gene therapy (VDEPT)). Also more than one prodrug activator can be used contemporarily (double transfection or use of a gene coding for a fusion protein consisting of 2 prodrug activators connected by a polyglycine spacer). E.g.

administered prodrug	transfected prodrug activator	produced drug(s)
CB1954 (5-aziridin-1-yl-2,4-dinitrobenzamide), SN24927	Escherichia coli aerobic or Bacillus amyloliquefaciens nitroreductase (NTR), mammalian Walker DT-diaphorase (DTD) [+ NAD(P)H]	2-hydroxylamine; 4-hydroxylamine ==non-enzymatic reaction with cellular thio-esters==> bifunctional alkylating agent
5-fluorocytosine (5-FC / 5-FCyt / flucytosine)	bacterial cytosine deaminase (bCD, usually from Escherichia coli)) or yeast CD (yCD, usually from Saccharomyces cerevisiae). CD is absent in mammalian cells.	toxic anabolite 5-fluorouracil (5-FU / 5-FUra) == cellular enzymes of rapidly proliferating (normal or tumor) cells ==> 5-fluoro-UTP (FUTP) and 5-fluoro-dUMP (FdUMP) : the former is incorporated into RNA and interferes with RNA processing, while the latter irreversibly inhibits thymidylate synthase and hence DNA synthesis. Tumour-selective amplification of the intracelular anabolic activation of 5-FU to FUMP can be achieved by transfection with the Escherichia coli gene for uracil phosphoribosyltransferase (UPRT)
6-methyl-9-(2-deoxy-b- D-erythro-pentofuranosyl) purine (6MEPDR)	Escherichia coli purine nucleoside phosphorylase (PNP)	6-methylpurine (6MEP)
fludarabine phosphate		2-fluoroadenine (2FA) : the compound can be incorporated into both RNA and DNA, killing non-dividing, as well as dividing cells. 2FA is 100-fold more potent as a growth inhibitor than 6MEP.
6-thioxanthine (6TX)	Escherichia coli XGPRT (gpt)
ganciclovir (GCV) (Cerepro®; source : Ark Therapeutics)	HHV-1 / HSV-1-tk (the equine herpes virus 4 tk is a better suicide gene than the HHV-1 tkref) : GCV phosphate transfer to non-HSV-TK-expressing bystander cells may mediate either bystander cell killing or sparing of HSV-TK-positive cells, depending upon the cell specific drug metabolismref.	GCV-monophosphate => cellular enzymes => GCV-triphosphate (kills dividing cells by acting as chain terminator)
acyclovir (ACV)		acyclovir monophosphate
tricyclic purine nucleoside analogues (TPNA) : lower affinity for HSV-tk; cytostatic potency and selectivity as for bicylclic compounds; more pronounced bystander effect in cell culture at relatively high drug concentrations; intrinsically strong fluorescent properties which allow simple and sensitive monitoring; higher lipophilicity => better uptake from the blood into CNS.  1) tricyclic ACV derivative 2) tricyclic GCV derivative
1-[3-(2-bromoethylamino)-2- hydroxypropyl]-2-nitroimidazole (RB6145) ==in vivo hydrolysis ==> 1-[3-aziridinyl-2-hydroxypropyl]- 2-nitroimidazole (RSU1069)	flavoprotein NADPH cytochrome P450 (c) reductase under the control of a hypoxia-responsive element (HRE) (O2-sensitive GDEPT)	highly reactive toxic species which can be back-oxidized to the non-toxic parent compound only in the presence of O2
cyclophosphamide (CP)	CYP2B6	4-OH-cyclophosphamide, that breaks imidazole ring in G leading to depurination of GMP residue.
indole-3-acetic acid (IAA)	horseradish peroxidase (HRP)
ethanol	alcohol dehydrogenase (ADH)	acetaldehyde (AcH)ref

Through cell membrane, gap junctions and/or following the death of the tumour cell, the cytotoxic drug may be able diffuse into neighboring cells through functional gap junctional intracellular communication (GJIC) and kill them (bystander effect, a biological amplification mechanism which is enhanced by contemporaneous administration of hydroxyurea (HU)), ensuring that it is not necessary to transfect all of the cells in the tumour. Fusion of TK to an 11-amino acid peptide from the basic domain of HIV-1 Tat protein (Tat11) imparts cell membrane-translocating ability to the enzyme and significantly increases its cytotoxic activity^{ref1, ref2}. The efficacy of this strategy was tested in 2 different mouse models of adoptive tumorigenesis, based on the implantation of human Kaposi sarcoma (KS-IMM) cells in nude mice or of B16F10 melanoma cells in syngeneic C57BL/6J mice. Experiments were performed by the subcutaneous injection of mixtures of unmodified tumor cells containing different fractions of TK or Tat11-TK producing cells followed by animal treatment with ganciclovir. In both systems mice bearing tumors containing Tat11-TK cells displayed significantly retarded tumor growth and prolonged survival as compared with mice inoculated with cells expressing unmodified TK. Collectively, these results demonstrate that fusion of Tat11 to TK imparts remarkable intercellular trafficking capability to the enzyme. This modification of TK might constitute an important step in the optimization of TK suicide gene strategy for gene therapy of cellular proliferation^ref
More efficient delivery of the prodrug across the blood-tissue barriers and maintaining continuous effective dose can be achieved by direct placement of a drug delivery system (e.g. silicone elastomer (Silastic^®) formulation) containing the prodrug. Fusion of TK to a cell-penetrating peptide (CPP) imparts cell membrane translocating ability to the enzyme and significantly increases its cytotoxic efficacy. In cells expressing CPP-TK, this protein is found extracellularly, associated with cell surface heparan sulfate proteoglycans (HSPG), and is released into the cell culture medium. Based on its interaction with HSPGs, the protein is then internalized by neighboring, nonexpressing cells, amplifying bystander effect by another route.
The susceptibility to killing may also be "preventive" (ie the drug may be administered later during life when need occurs) : eg. introduction into stem cells before transplantation might provide a means of permitting the ablation of these cells if they subsequently misbehave (malignancy in any tissue that retains growth potential, immune pathology on the part of lymphocytes (e.g. donor T cells for GvHD, ...), or hyperfunction on the part of cells intended to supply a missing function at regulated levels). Particular concerns may apply to ESCs because of evidence of their epigenetic instability. Ionizing radiation (IR)-responsive promoters can be used to combine radiotherapy and gene therapy.
• indirect killing
• tumor amplified protein expression therapy (TAPET)
• mesenchymal stem cells (MSCs) genetically engineered with AAV vector are attracted by tumors as they use these cells for tumour neoangiogenesis (just like they migrate to injuries to repair wounds) and continue to develop and grow in the tumor environment, representing an effective delivery system for gene therapy in pediatric soft-tissue sarcomas
• bacterial GDEPT : systemically delivered non-pathogenetic, genetically engineered obligate anaerobic Bacteria that exclusively germinate and replicate in hypoxic/necrotic regions after systemic or intratumoral application (e.g. solid cancers)
• bacteria releasing their contents nearby
• spores of
• Clostridium butyricum M-55 (Mose strain Cl. oncolyticum M55)
• Clostridium beijerinckii^{ref1, ref2}
• Clostridium sporogenes^ref
• Bifidobacterium adolescentis
• Bifidobacterium longum, which is nonpathogenic and anaerobic
• Salmonella typhimurium strain VNP20009 is a genetically modified strain possessing an excellent safety profile, including genetically stable attenuated virulence (a deletion in the purI gene), reduction of septic shock potential (a deletion in the msbB gene), and antibiotic susceptibility. The combination of the lipid mutation and the purine auxotrophy attenuate the virulence of the bacteria by greater than 10,000-fold and enhance the specificity of the bacteria for tumor tissue (accumulate in tumor tissue at levels at least 200-fold and, more commonly, 1000-fold greater than in other normal tissues : colonization of normal tissues gradually decreases with time^ref). In mice, VNP20009 is rapidly cleared from the blood from a peak level of 1x10⁴ cfu/mL to undetectable levels in 24 h. In nonhuman primates, VNP20009 was also rapidly cleared from the blood, from a peak level of 1.0x10⁶ CFUs/mL to undetectable levels in 24 h. VNP20009 was detected in the liver, spleen, and bone marrow of monkeys; the amount decreased over time, and VNP20009 was cleared from all organs by day 41; no VNP20009 could be detected in the urine or feces of the monkeys. VNP20009 is genetically stable after many generations of growth (> 140) both in vitro and in vivo^ref. These bacteria have been found to be safe in mice, pigs and monkeys when administered intravenously. 4-5 days after a single IV injection of 1 x 10⁶ CFUs/mouse, we routinely detected VNP20009 proliferation and accumulation at levels ranging from 1 x 10⁸ to 2 x 10⁹ CFUs/g tumor. The amount of VNP20009 accumulated in the liver ranged from 3 x 10⁴ to 2 x 10⁶ CFUs/g. The distribution of Salmonella in tumors was homogenous; YS7212 could be detected from the periphery to the interior portion of the tumors. Bacteria accumulation and CD expression persisted in the tumors for up to 14 days after a single bolus IV administration of bacteria to tumor-bearing mice^ref. Optimal therapeutic cose (OTD) was found to be 5 x 10⁷ CFUs/rat. In humans by the intratumoral route, a maximum tolerated dose has not been reached, and dose escalation continues past the current dose level of 4 x 10⁷/m². Furthermore, VNP20009 persisted in injected tumors for at least 2 weeks in 8/11 patients treated to date. By 30-min IV administration, a maximum tolerated dose (MTD) of 3 X 10⁸ CFUs/m² has been established. The maximum-tolerated dose was 3 x 10⁸ CFUs/m². Dose-limiting toxicity was observed in patients receiving 1 x 10⁹ CFUs/m², which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as IL-1b, TNF-a, IL-6, and IL-12. Focal tumor colonization was observed in two patients receiving 1 x 10⁹ CFUs/m² and in one patient receiving 3 x 10⁸ CFUs/m^2ref. In all patients treated to date, VNP20009 was not shed in urine or stool^ref.
• expressing HSV1-TK (imaging with [¹⁴C]FIAU)
• VNP20047
• VNP20029 (chromosomal insertion of an E. coli cytosine deaminase (CD) gene at the DmsbB locus : TAPET-CD) allows tumor growth inhibition of >90% in several murine tumor models. When 5-FC was administered by intraperitoneal (IP) injection once or 3 times daily to tumor-bearing mice that had been pre-treated with TAPET-CD, high levels of 5-FU (reaching 20-40 mM/g) were detected in the tumor, with low or undetectable 5-FU levels in normal tissues (e.g., spleen, liver, etc.)^ref
• YS7212
• bacteria entering the actual tumour cells
• taking advantage of the proximity of bowel mucosa to luminal bacteria, attempts to deliver a therapeutic gene to the colonic mucosa have been performed by oral administration of an invasive and non-pathogenic Escherichia coli diamenopimelate (dap) auxotroph, harboring plasmid pGB2Winv-hly which express the invgene from Yersinia pseudotubercolosis that confers the ability to invade nonprofessional phagocytic cells and the hly gene from Listeria monocytogenes that allows expression of lysteriolysin O, a perforin cytolysin able to perfore phagosomal membranes. This bacterial vector invades and transfers functional DNA to epithelial cells in vitro. This strain may be loaded with
• a chemotherapeutic drug : listeriolysin O helps the bugs spill their contents, releasing the drug-activating enzyme : > 90% of cells invaded by bacteria were killed by 6MEDPR, compared with < 15% of non-invaded cells^ref
• another therapeutic gene :
• pC1WTGF-b₁ can significantly reduce the severity of experimental colitis in mice. However, as a consequence of mucosal barrier disruption during colitis, vector-specific mRNA transcripts could be recovered from the colon and also from extra-colonic tissues. The constitutive CMV promoter in pC1WTGF-b₁ was therefore replaced by the inflammation-inducible IL-8 promoter generating plasmid pC1WTGF-b_1IND. Plasmid-specific TGF-b₁ mRNA transcripts were detectable in mouse CMT-93 epithelial cells incubated with E. coli BM2710/pGB2Winv-hly carrying pC1WTGF-b_1IND following exposure to inflammatory cytokines. Furthermore, the transcripts were detectable only within inflamed tissues and the therapeutic effects were comparable to those in animals treated with E. coli BM2710/pGB2Winv-hly+pC1WTGF-b₁^ref.
• a satellite DNA cloned in P1-based artificial chromosome (PAC) was stably delivered into the HT1080 cell line and efficiently generated human artificial chromosomes de novo. Similarly, a large genomic CFTR construct of 160 kb containing a portion of the CFTR gene was stably propagated in the bacterial vector and transferred into HT1080 cells where it was transcribed, and correctly spliced, indicating transfer of an intact and functional locus of at least 80 kb. These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis. Such an approach opens new perspectives for gene therapy^ref.
• Mycobacterium bovis bacillus Calmette-Guerin (BCG) locally injected for carcinoma in situ of the bladder expressing
• mouse IL-18 augments T_h1 immunity and macrophage cytotoxicity^ref
• TNF-a
• by immune system cells, transfecting genes coding for immunostimulatory molecules (immunogenes / immuno-gene therapy) ...
• ... into normal immune system cells (this is a sort of adoptive immunotherapy !)
• secretion gene therapy (SGT)
• activatory cytokines in lymphocytes (e.g. IL-4 or TNF-a in TILs : when reinjected in bloodstream they spontaneously tend to migrate into the tumor mass)
• receptors for activatory cytokines
• gene transfer of both CD80 and CD154 by lentiviral vectors was necessary to significantly improve the APC function of human autologous primary multiple myeloma (MM) cells. Simultaneous CD80/CD154 expression on MM cells allowed the generation of CD8⁺ T cells that recognized unmodified MM cells in 11 of 16 cases, specifically in 6 of 6 patients with low-stage disease, but only in 5 of 10 patients with advanced disease. The activity of CD8⁺ T cells was MHC restricted and MM specific. In 7 of 7 cases, CD8⁺ T cell activity was inhibited by mAbs against HLA class I, and in 4 of 4 cases, CD8⁺ T cells recognized autologous MM cells but not autologous normal B and T lymphocytes nor bone marrow stromal cells. In addition, the activity of CD8⁺ T cells was directed against allogeneic MM cells that shared at least one MHC allele with the autologous counterpart, but not against MHC mismatched MM cells. These data lay the ground for the isolation of new MM antigens and for the design of vaccination protocols with primary MM cells genetically engineered to express immunostimulatory molecules^ref.
• non self Ag + activatory cytokines in APCs (e.g. tumor Ag + IL-2, IL-4 and IFN-g  in BMDCs)
• generating T lymphocytes possessing an antigen specificity in addition to that encoded by their constitutive TcR. Generally chimeric TcR are fusion proteins consisting of a MHC-non restricted antigen specific scFv joined to a signaling chain (g, z, ...) capable of initiating T cell activation (ie cytokine release and target cell lysis).
• immunogen and non-immunogenic tumor-associated antigen
• a bicistronic retroviral vector was engineered that efficiently delivers genes for both a and b chains of TcR to HSCs for antigen-specific adoptive immunotherapy. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8⁺ or CD4⁺ T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TcRs specific for a model tumor antigen, the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8⁺- and CD4⁺-specific TcRs, and boosting with DCs pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens^ref.
The immune function of retrovirus-mediated gene modified T cells is critical for a beneficial effect to follow their adoptive transfer into patients. The majority of retroviral vectors used in this therapeutic strategy are derived from mammalian C-type murine leukemia viruses (MLVs), and infect only dividing cells. Therefore, ex vivo expansion of the target cells is required for efficient gene transfer with these vectors. The process of ex vivo expansion may alter the function of gene modified T cells. In fact, there is evidence to suggest that the expansion process rather than the integration or expression of the transgene may have the largest effect on the function of gene-modified T cells in vitro and in animal models. While CD3/CD28 stimulation parallels the lineage differentiation patterns induced by antigens in physiological conditions, PHA induces a skewed distribution of the memory T cell subsets, with near disappearance of the subpopulations that display the effector memory phenotype in favour of the central memory subpopulation.
• ... into normal surrounding cells
• secretion gene therapy (SGT)
• scFv specific for antigens of the targeted cells
• activatory cytokines (e.g. IL-2 and IL-4 in fibroblasts) or chemokines : they can provoke nonspecific inflammatory cascade.
• expression of addressins on vascular endothelium
• ... into targeted cells (ex vivo only when they are culturable; with plasmid electroporation^ref; if cancer cells are transfected : transfectoma)
• secretion gene therapy (SGT)
• activatory cytokines, eg.
• IGF-1 antisense
• IFN-a
• IFN-g
• TNF-a
• IL-2
• IL-4
• IL-6
• IL-7
• CCL2 / JE
• GM-CSF
• G-CSF
• TNF-a with ionizing radiation (IR)-responsive promoter (TNFerade^® from GenVec Inc.)
• Fv of 4-1BB-specific antibody : melanoma^ref (CD4 T-cell response augmented)
• surface molecules, eg.
• CD80 / B7.1 & CD86 / B.7.2
• membrane-bound GM-CSF (mbGM-CSF)
• LIGHT : breast carcinoma^ref (CD8/CD4 T-cell response augmented ?) and mastocytoma^ref (CD8 and CD4 T-cell response augmented)
• CD27 L / CD70 : sarcoma^ref (CD8/CD4 T-cell response augmented ?), mastocytoma^ref (CD8 T-cell response augmented) and adenocarcinoma^ref (CD8/CD4 T-cell response augmented ?)
• OX40L : colon carcinoma^ref (CD8 and CD4 T-cell response augmented)
• CD137L / 4-1BBL : sarcoma, mastocytoma^ref, lymphoma^ref and squamous cell carcinoma^ref (CD8 T-cell response augmented)
• non self Ags (e.g. HLA-B7, b₂m)
• ... into whole body
• secretion gene therapy (SGT)
• activatory cytokines for DCs hematopoiesis (FLT3L)
Because immunity is a systemic reaction, this immune reaction could potentially eliminate all the tumour cells in the body (biological amplification mechanism !), including sites of metastatic deposit.
• non conventional gene therapy
• correcting the mutated allele (gene repair) : it is expecially effective against dominant negative mutations (e.g. X-SCID) and genes whose expression requires strict control by thier normal promoters (e.g. HIGM1)
• inhibiting the expression of a gene ("antigene" strategies) : it is expecially effective against dominant negative alleles, mutations which cause inappropriate expression patterns and/or acquisition of new functions (e.g. oncogenes), genes of infectious agents.
• at DNA level
• triplex forming oligonucleotides (TFO) or double-stranded oligodeoxynucleotides (dsODN) as a decoy corresponding to the cis sequence 
• human papillomaviruses E2 protein is able to regulate negatively the expression of E6 and E7 papilloma oncoproteins in cervical carcinoma.
• at RNA level
• 2 companies—Acuity Pharmaceuticals in August and Sirna Therapeutics in September 2004—filed applications with the US Food and Drug Administration to begin testing the technique's ability to treat age-related macular degeneration by interfering with the growth of new blood vessels
• see below for hypercholesterolemia
• inhibiting the function of the protein, by transfecting a gene encoding for ...
• adaptamer or aptamer
• intracellular antibodies / intrabody
• genes coding for transdominant negative proteins
• catalytically inactive polymer subunit (e.g. ErbB2 kinase dead form)
• receptor decoys
• intracellular receptor decoys (e.g. CCL5 / RANTESintrakine for CCR5 : an intrakine is a modified cytokine with the KDEL ER retention signal that prevents secretion. The intrakine binds to the newly synthetised CCR5 molecules and traps them in the ER, where they are degraded. RANTES-intrakine expressing primary CD4⁺ T lymphocytes were found to resist M-tropic HIV-1 infection, while maintaining basal biological features).
• soluble receptor decoys (e.g. sCD4 for HIV-1 gp120)
• adding another gene that can make up for the function of the mutated gene. E.g. :
• utrophin in DMD
• HbF (in b-thalassemia and SCA)
• silencing the expression of a normal gene which contributes to the pathogenesis of the disease. E.g. :
• BcR-associated protein (BAP) 31, chaperone proteins, mucins or the epithelial sodium channel (ENaC) in CF.
• adding anti-apoptotic genes to rescue cells that are undergoing apoptosis
• p35 from Baculovirus inhibits the activation of caspases 1, 3, 6, 7, 8, and 10
• g34.5 from the HSV dephosphorylates eIF2a and inhibits the shutoff of protein synthesis in metabolically-stressed host cells
• crmA from cowpox virus inhibits the activation of caspases 1 and 8 and prevents IL-1b and IL-18 processing.
• KsBcl-2, a HHV-8 homolog of Bcl-2 which lacks a loop domain and has poorly conserved BH3 and BH4 domains, escaping negative regulation by pro-apoptotic kinases and caspases.
• selection of a given population which has co-undergone gene therapy
• conferring resistance to negative selection (indirect proliferative advantage)
• resistance to a drug then administered to kill untransfected cells
• ABCB1 / P-glycoprotein (P-GP) / MDR1 and paclitaxel
• O⁶-alkylguanine-DNA-alkyltransferase and alkylating agents
• mutant dihydrofolate reductase (DHFR) and trimetrexate
• resistance to viral infections
• CCR5siRNAs in T lymphocytes against HIV-1 infection
• conferring advantage to positive selection (direct proliferative advantage) : selective amplified genes (SAG) or survival switches or cell growth switches. Activation of the transenzyme involved in a survival (usually anti-apoptotic) signal transduction pathway can be triggered as described here.
• combined immunotherapy including gene therapy and DC vaccines may have some advantages in combating well-established tumors vis-a-vis either modality administered as a monotherapy^ref

The UK leads Europe in this field with 40% of European gene therapy clinical trials being carried out in the UK. Since approving the first gene therapy trial in the UK in 1993, the Gene Therapy Advisory Committee (GTAC) has approved 96 gene therapy clinical trials, involving almost 900 patients. These gene therapy studies target inherited disorders, infectious diseases such as HIV infection, and cardiovascular disease. The majority (71%) of trials are for the treatment of cancer, including breast, ovarian, cervical, pancreatic, prostate, bladder, head & neck, blood, colorectal and liver cancer as well as skin cancer.
The vast majority of gene therapy clinical trials have been phase I clinical trials. By the end of 1996 to 2002 only 2 gene therapy trials had progressed to a phase II stage. To date no one has been cured through the use of gene therapy and there has been little evidence of therapeutic efficacy in most gene therapy protocols. However, the aims of most gene therapy clinical trials have been less ambitious and have sought mainly to demonstrate detectable gene expression in the target tissues.
A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to most muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Inclusion of VEGF/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced transduction of both cardiac and skeletal muscles at lower vector doses after a single intravenous administration of recombinant AAV pseudotype 6 vectors^ref
Web resources :
• Gene therapy clinical trials worldwide
• ClinicalTrials.gov
• diseases with intrinsic aetiology : in the future, the potential use of stem cell-derived cell platforms with gene transfer might allow for a maximally beneficial approach to regenerative medicine^ref. In the stem cell context, one must consider that autologous. stem cells may be prone to recurrence of the underlying disease that led to end-organ damage originally. Meanwhile, the use of allogeneic stem cells will entail immune suppression just as it does in solid organ transplantation. Only through gene-modification of autologous stem cells can the full potential of regenerative medicine be realized.
• cystic fibrosis (CF) (18 trials have been carried out at October 2002) :
• spray-mediated transfection of the gene encoding CFTR into airway epithelial cells. Transfection efficiency could be increased through pre-treatment with mucolytics, recombinant DNase or the anticholinergic drug glycopyrolate. Receptors for Ad and AAV2 are located on the basolateral membrane of human airway epithelial cells : anyway the transient opening of tight junctions (by EGTA, anti-E-cadherin antibody, sodium caprate, a blend of sucrose, mannitol and Pluronic F68 or perfluorochemical) is clinically unapplicable given that the airways of most CF patients are heavily colonised with bacteria
• several new recombinant viruses have been identified, that appear to be able to enter epithelial cells via the apical membrane efficiently, e.g.RSV, AAV5 and Sendai virus (SeV)
• alternatively Ad and AAV2 vectors can be retargeted to apical surface receptors (B₂, uPAR, P2Y₂)
• up to 3 monthly aerosol inhalations (each nominal dose is 10¹² particles) of AAV2 vector expressing CFTR (tgAAVCF) were safe and well tolerated, and resulted in encouraging trends in improvement in FEV₁ and reduction in inflammation (IL-8) in the sputum of patients with mild CF (FEV₁ at least 60% predicted). All subjects receiving tgAAVCF exhibited an increase (by at least 4-fold) in serum AAV2-neutralizing antibodies and detectable levels in BAL fluid from 5 of 6 treated subjects undergoing BAL. Gene transfer but not gene expression was detected in a subset of 6 tgAAVCF subjects who underwent bronchoscopy^ref
• enveloped viruses (expecially lentiviral vectors) can be pseudotyped with glycoproteins from viruses that naturally infect the airway epithelial cells, such as human coronavirus 229E, influenzavirus A strains and members of the Filoviridae family
Intravenous administration and in utero somatic gene therapy are under investigation.
• ornithine transcarbamylase (OTC) deficiency : systemically transfected hepatocytes with Ad vectors
• hemophilia A :
• Requinate^® (source : Oxford BioMedica plc) consists of a LentiVector system carrying the Factor VIII gene. It is administered to the liver via intravenous injection
• hematopoietic stem cell (HSC)–directed gene therapy with a retroviral vector expressing ... :
• human fVIII gene^ref
• porcine fVIII cured hemophilia A mice without inhibitor development^ref. There are 2 major advantages to using the porcine gene. First, the porcine fVIII gene resulted in greater activity in vivo than the human fVIII gene, which is probably due to a higher specific activity of the pig protein. Second, most inhibitors to human fVIII do not cross-react with porcine fVIII, making it possible that a vector with the porcine gene might be used in the large subset of patients that already has inhibitors to human fVIII. Although plasma-derived porcine fVIII is not currently available, recombinant porcine fVIII is being tested^ref. There are also caveats. First, use of this gene required at least partial bone marrow ablation, which has toxicities, and would be difficult to justify for patients with hemophilia. However, an in vivo delivery of retroviral vector to HSCs could have a similar effect. Indeed, neonatal intravenous injection of a retroviral vector expressing canine fVIII cured hemophilia A dogs without inhibitor formation^ref. Since this procedure^ref transduces HSCs in addition to liver cells, it is possible that the tolerance to canine fVIII was due to the transduction of blood cells. An in vivo delivery without selection should be clinically acceptable if it were safe. However, the second caveat of using HSC-directed gene therapy in patients is that an integrating retroviral vector might result in leukemia due to insertional mutagenesis
• hemophilia B : the risk of an immune response to the coagulation factor IX (F.IX) transgene product is a concern. In order to investigate the mechanism of F.IX-specific lymphocyte activation in the context of AAV gene transfer to skeletal muscle, AAV-2 vector expressing human F.IX (hF.IX) was injected into outbred immune-competent mice. Systemic hF.IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4+ T and B cells. ELISPOT assays documented robust responses to hF.IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF.IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX^ref.
• a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B1. A phase 1/2 dose-escalation clinical study was carried out to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10¹² vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression^ref.
• a protocol for reducing inhibitor formation in gene therapy by prior mucosal (intranasal) administration of a peptide representing a human F.IX-specific CD4⁺ T cell epitope in hemophilia B mice. C3H/HeJ mice with a F.IX gene deletion produced inhibitory IgG to human F.IX after hepatic gene transfer with an AAV vector. These animals subsequently lost systemic F.IX expression. In contrast, repeated intranasal administration of the specific peptide resulted in reduced inhibitor formation, sustained circulating F.IX levels, and sustained partial correction of coagulation following hepatic gene transfer. This was achieved through immune deviation to a T_h cell response with increased IL-10 and TGF-b production and activation of regulatory CD4⁺CD25⁺ T cells^ref
• gene transfer to hematopoietic stem cells (HSCs)
• gene marking : using retroviral vectors to insert biologically inert reporter genes into HSCs or cancer cells allows for in vivo tracking of transplanted cells.
• chemotherapy resistance
• HIV-1 resistance
• gene replacement for genetic diseases
• haemoglobinopathies and other erythrocyte defects
• b-thalassemia : b-globin
• sickle-cell anemia (SCA) : b-like globin
• G6PD deficiency : G6PD
• porphyria : ferrochelatase, others
• cytoskeletal defects : spectrin b, erythrocytic
• immunodeficiencies
• X-linked immunodeficiency with hyper-IgM (HIGM1) : as constitutive expression of CD40L induces lymphomas, the mutation needs to be corrected while preserving the natural regulation of CD40L using pre-mRNA trans-splicing (spliceosome-mediated trans-splicing (SMaRT)). Bone marrow from mice lacking CD40L was modified with a lentivirus trans-splicer encoding the normal CD40L exons 2–5 and was administered to syngenic CD40L-knockout mice. Recipient mice had corrected CD40L mRNA, antigen-specific IgG₁ responses to KLH immunization, regulated CD4⁺ T-cell CD40L expression after CD3 stimulation in primary and secondary transplanted mice, attenuation of Pneumocystis carinii pneumonia, and no evidence of lymphoproliferative disease over 1 year^ref
• Wiskott-Aldrich syndrome (WAS) : WASp in HSCs.
• SCIDs
• ADA-SCID : T-cells are isolated from blood and stimulated to proliferate in vitro, before being transduced with a retroviral vector containing the ADA gene. The cells are then culture-expanded and re-infused into the patients. The first approved human gene therapy trial was led by Dr William French Anderson, R. Michael Blaese, and Kenneth Culver in September 1990 at the University of Southern California on a 4-year-old girl, Ashanthi de Silva. The girl continues to do well but it is unclear whether that is due to the therapy because she is also on a low-dose regimen of intravenous synthetic ADA therapy, her immune system is now fully functional, with 20% to 25% of all her T cells containing the gene that was introduced by retroviral transfer^ref

[ Anderson, the director of the Gene Therapy Laboratories at the University of Southern California's Keck School of Medicine in Los Angeles and a professor of molecular biology, biochemistry, and pediatrics, was arrested on July 30 Friday morning as he was leaving for work and charged with 6 counts of sexual child molestation. After spending the weekend in jail, Anderson was released after posting $600,000 bail. On Mon August 2, Anderson pleaded not guilty to all charges. He was asked to reappear in court August 16 to schedule a date for a preliminary hearing. The alleged victim was Anderson's student in Karate who is now 17 years old. The alleged molestation occurred between 1997 (when the girl was 10) and 2001 in Anderson's home in San Marino, Calif. Prosecutors in Maryland said on Wed Aug 7 2005 they dismissed the case against William French Anderson less than a week before trial because key evidence against him was inadmissible under state law. The inadmissible evidence was a recorded telephone call between Anderson and his Maryland accuser in which the scientist discussed the Los Angeles County case against him. Unlike California, Maryland does not allow evidence of similar allegations to be presented in sex cases. However, the Maryland accuser could testify in the California case. The Maryland accuser claimed Anderson molested him during martial arts lessons from 1983 to 1985, beginning when he was 12. Anderson's California trial likely won't start until at least January 2006. If convicted on all counts, the scientist, 67, could spend up to 56 years in jail. Anderson's home and office have been searched, and some computers have been removed from his office as part of the case. Anderson has focused his recent research on gene therapy delivery systems. Specifically, he was working to design virus-based vectors to transfer genes and better vectors to insert genes into hematopoietic stem cells. He has also focused his efforts on developing genetic therapy techniques that can be used in utero, and nanotechnology for genomic applications, such as creating a nanochip that builds a cDNA library using only one cell. Before joining the University of Southern California, Anderson spent 27 years as a gene therapy researcher at the National Heart, Lung, and Blood Institute at the National Institutes of Health. To date, Anderson has published almost 400 research articles and been profiled by a number of publications, including the New York Times and Scientific American. Anderson is founding editor of the journal Human Gene Therapy and was editor-in-chief until 2003. He also has a fifth degree black belt in Tae Kwon Do, and continues to compete in karate competitions.  ]
• Milan, Italy
• Wolfson Centre for Gene Therapy of Childhood Disease at the Institute of Child Health in London launched the ADA trial in 2003 : Mustaf, born in September 2000, was chosen because a marrow donor with a good tissue match could not be found and he did not respond well to PEG-ADA
• X-SCID : CD132 / IL-2Rg_c
• ex vivo transduction of HSCs with CD132 / IL-2Rg_c-encoding MLV. The cells are then culture-expanded and re-infused into the patients. The clinical trial led by Professors Marina Cavazzana-Calvo and Alain Fischer at the Hôpital Necker-Enfants Malades in Paris, France, involving MoMLV-mediated expression of g_c in ex vivo-transfected HSCs of 11 X-linked SCID patients from 5 countries (successful in 9 patients), was suspended in October 2002 as in a 3-year-old boy retroviral integration inside LMO2 oncosuppressor gene (which encodes a transcription factor required for hematopoiesis) had caused g/d T cell leukemia, first apparent in August, 30 months after the gene therapy. Another boy fell ill on January 2003 due to integration near enough to LMO2 gene, too. These patients were the youngest to be treated—just 1 and 3 months—while the others were all at least 6 months old. They also received the highest number of corrected cells : so, statistically speaking, the more you inject, the higher the probability. Anyway gene therapy has a relatively good risk–benefit ratio : all 14 children treated to date are alive and well, and the 2 boys who developed leukemia responded well to chemotherapy and are in complete clinical remission^ref. A third, 3-years old, boy developed leukemia and died in February 2005 : FDA suspended trials in USA. 7 children and 1 adult have been treated at Great Ormond Street Hospital in London.

Factors that underlie the development of leukemia in patients with X-linked SCID :
• insertional oncogenesis : activation of cellular proto-oncogene; inactivation of tumour-suppressor gene
• effects of transgene : cDNA expression facilitates LMO2-mediated transformation^ref
• high percentage of bone-marrow stem/progenitor cells that are susceptible to retroviral-mediated transduction : intrinsically high haematopoietic stem/progenitor cell content in bone marrow of infants; X-linked SCID-related increased proliferative activity of stem/progenitor cells
• LMO2 is an accessible integration target due to its expression in HSCs : preference for retroviral integration at transcriptionally active chromosomal regions
• nature of X-linked SCID : high selective survival advantage of corrected cells increases seeding efficiency; high proliferation of transduced T-lymphoid progenitors facilitates LMO2-mediated transformation; immune deficiency allows transformed cells to escape immune surveillance
Using a mouse model for gene therapy of X-SCID, we find that the corrective therapeutic gene IL2RG itself can act as a contributor to the genesis of T-cell lymphomas, with one-third of animals being affected. Gene-therapy trials for X-SCID, which have been based on the assumption that IL2RG is minimally oncogenic, may therefore pose some risk to patients^ref.
• using engineered zinc-finger nucleases—DNA-binding proteins that target specific sequences—researchers at Richmond, Ca.–based Sangamo Biosciences corrected a defective version of the CD132 / IL-2Rg_c receptor gene, which is mutant in children with X-SCID, achieving the highest levels of permanent targeted gene correction in cultured human cells : > 18% of human immune cells were correctly modified without selection, of which 7% acquired the desired modification in both chromosomes (recent estimates have found an absolute rate of gene targeting in human cells of 3 to 5%^ref). The technique cannot currently be used to correct cells within the body, but the researchers think they could extract stem cells, which give rise to the affected immune cells, correct their mutations, and put them back into the patient's body to restock the immune system. Sangamo had used zinc-finger proteins before in several instances, among them, to inhibit specific single gene expression in vivo. The key for the researchers' success was their ability to introduce a break in the desired gene : normally, gene targeting is done by isolating and manipulating a segment of the gene in the laboratory, and then putting it back into cells, hoping that homologous recombination will lead to the replacement of the endogenous gene with this manipulated gene. The problem is that the frequency of that sort of gene targeting is typically quite low—about 1 in 1 million cells gets the targeting event. Sangamo's strategy built upon 2 fundamental observations previously made by other researchers: that a DSB stimulates HR at a target and that such breaks can be made with designed zinc-finger nucleases. Although other researchers had been successful at targeting specific sequences, no one had designed the zinc-finger protein nucleases from scratch. The zinc finger protein was able to comb through the millions of letters in the human DNA code to find a single 24-letter sequence within the defective gene that causes SCID. They do this by mixing and matching an array of proteins that recognize and bind to specific 3-letter sequences of DNA : by sandwiching together eight of these proteins, for example, they can design one that binds to a chosen 24-letter sequence. They fused this protein to an enzyme that snips through DNA at that site. The researchers aimed to insert this enzyme into human cells, along with a fresh piece of DNA that matched the defective gene but lacked the mutation. Severing the DNA triggers a repair process called recombination, in which the cell attempts to patch up a cut by swapping the entire region with an undamaged segment, in this case, the unmutated DNA sequence.  Researchers at Sangamo are also developing their technique to tackle HIV. Rather than correct a mutation, the idea is to introduce a defect into a gene in immune cells, so that they no longer allow the virus to enter. Indeed, similar enzymes can be designed that recognize and cut DNA at any point in the human genome, potentially triggering almost any change or repair. The gene-correction technique ought to avoid the integration problem, because the new DNA replaces the old copy at the same site, but it may, for example, prove difficult to engineer enzymes that recognize particular spots in the genome. And there are concerns that the enzyme might snip and damage DNA at an inappropriate place
• JAK3
• leukocyte defects :
• chronic granulomatous disease (CGD) : gp91^phox, p47^phox
• leukocyte adhesion defect (LAD) : CD18
• leukodystrophies :
• X-adrenoleukodystrophy : ALDP
• metachromatic leukodystrophy : arylsulfatase A (ARSA) / cerebroside sulfatase
• lysosomal storage disease (LSD)
• Gaucher's disease : acid b-glucosidase /b-glucocerebrosidase / glucosylceramidase
• mucopolysaccharidosis type IH (MPS IH) / Hurler's syndrome : a-L-iduronidase
• stem-cell defects :
• Fanconi's anaemia : FANCA, FANCC, FANCE, FANCF, or FANCG, or FANCD2
• familial hypercholesterolemia (FH) might be treated by transferring the gene encoding for the LDL-R into hepatocytes in order to reduce the level of serum cholesterol.
• muscle atrophy : IGF-1 targeting to rat muscles induces growth in size and strength by 15 to 30% (when they also put through an exercise program, the animals double their muscle strength).
• Duchenne's muscular dystropy (DMD) : most effort to replace the defective dystrophin gene has concerned an ex vivo strategy to transfect isolated muscle cells with multiple copies of the dystrophin gene and re-implant the cells back into the patient’s muscle. There has so far been little work on the in vivo delivery of the dystrophin gene as there are few vectors suitable for the efficient delivery of genes into muscle cells in vivo and ensure tissue specific expression, although AAV can deliver microdystrophin^ref. Systemic gene delivery into muscle has been a major challenge for muscular dystrophy gene therapy, with capillary blood vessels posing the principle barrier and limiting vector dissemination. Previous efforts to deliver genes into multiple muscles have relied on isolated vessel perfusion or pharmacological interventions to enforce broad vector distribution. The efficiency of multiple AAV vectors was compared after a single injection via intraperitoneal or intravenous routes without additional intervention. AAV8 is the most efficient vector for crossing the blood vessel barrier to attain systemic gene transfer in both skeletal and cardiac muscles of mice and hamsters. Serotypes such as AAV1 and AAV6, which demonstrate robust infection in skeletal muscle cells, were less effective in crossing the blood vessel barrier. Gene expression persisted in muscle and heart, but diminished in tissues undergoing rapid cell division, such as neonatal liver^ref.
• antisense oligonucleotides to the boundary sequences of exon and intron 23 of the dystrophin gene in the drug delivery agent F127 result in successful splicing-out of the mutant exon and production of an in-frame smaller dystrophin protein. The level of the shortened dystrophin molecule peak 2 to 4 weeks after injection and persist for over 2 months. Following a second injection after 3 months, additional muscle fibers are observed to express the dystrophin protein: the administrations do not induce an immune response or a diminution of efficiency with age
• utrophin can make up for the function of dystrophin
• congenital muscular dystrophy 1D (MDC1D) : LARGE glycosyltransferase overexpression can functionally bypass a-dystroglycan (a-DG) hypoglycosylation and marked reduction of its receptor function in Large^myd mice^ref
• X-linked ichthyosis: direct injection of naked DNA coding for transglutaminase 1 (TGM1) in keratinocytes.
• junctional epidermolysis bullosa : injection of naked DNA coding for laminin b₃ in keratinocytes.
• Crigler-Najjar syndrome: helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. A HD-Ad vector has been used to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to <<1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 x 10¹² viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after 1 injection of vector, and i.v. injection of bilirubins IIIa and XIIIa in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 x 10¹¹ viral particles per kg. Complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity^ref.
• glycogen storage disease (GSD)
• type Ia GSD (von Gierke's disease) : catalytic subunit of G6Pase.
• type II GSD: acid a-1,4-glucosidase / acid maltase^ref
• lysosomal storage disease (LSD)
• mucopolysaccharidosis type VII (MPS VII) : normal NSCs are engineered to overexpress the normal secreted enzyme b-glucuronidase (GUSB) for ex vivo secretion gene therapy, providing a source of normal enzyme for uptake by diseased cells (cross-correction).
• diseases with extrinsic aetiology
• viral infections
• HIV-1
• confering resistance to negative selection by viral infection
• dominant negative (dn) HIV-1 genes
• RevM10
• Dgag (interference with protein oligomerization)
• antisense therapy
• trecovirsen : a 25-mer antisense phosphorothioate oligonucleotide targeted at the gag gene
• VRX496 (source : VIRxSYS Co.) : autologous CD4⁺ T cells are isolated from patients and infected with a VSV-G-pseudotyped HIV-based vector that encodes an antisense payload against the HIV env gene. The antisense payload is under the control of the native LTR promoter, which is highly transactivated by tat upon HIV infection in the cell. T cells containing the integrated viral genome (VRX496T) are re-infused into patients so that when HIV tries to infect the modified T cells in vivo, the anti-sense mRNA is produced and binds to the sense viral mRNA, thereby preventing translation of viral proteins. no adverse events have been observed in 3 patients : viral load results are not above pre-dose levels and CD4 counts have remained stable. This is the first time that a lentiviral vector has been used in a clinical trial in humans, and concerns about recombination and other safety issues have led many researchers to abandon research^{ref1, ref2, ref3}
• antisense ribozymes
• an inducible anti-HIV short hairpin RNA (shRNA) expressed from a Pol II promoter inhibits HIV-1 gene expression in mammalian cells. The strategy is based on a promoter system in which the HIV-1 LTR is fused to the Drosophila hsp70 minimal heat shock promoter. This system is inducible by HIV-1 TAT, which functions in a negative feedback loop to activate transcription of an shRNA directed against HIV-1 rev. Upon induction the shRNA is processed to an siRNA that guides inhibition of HIV replication in cultured T-lymphocytes and HSC–derived monocytes. The fusion promoter system may be safer than drug-inducible systems for shRNA-mediated gene therapy against HIV as the shRNAs are only expressed following HIV infection^ref
• RRE and TAR decoys for Rev and Tat, respectively
• intrabodies to a-Tat
• suicide genes under LTR
• TK
• diphtheria toxin a
• genetically engineered live microbes : most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. Such naturally occurring bacteria can be genetically engineered to protect against HIV infection by secreting antiviral peptides. Nissle 1917 is a highly colonizing probiotic strain of Escherichia coli secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides were achieved. The genetically engineered Nissle 1917 were capable of colonizing mice for periods of weeks to months, predominantly in the colon and cecum, with lower concentrations of bacteria present in the rectum, vagina, and small intestine. Histological and immunocytochemical examination of the colon revealed bacterial growth and peptide secretion throughout the luminal mucosa and in association with epithelial surfaces. The use of genetically engineered live microbes as anti-HIV microbicides has important potential advantages in economy, efficacy, and durability^ref.
• ImmStat^® (source : Oxford BioMedica plc) consists of anti-HIV therapeutic genes designed to disrupt the viral infection and replication stage. Gene delivery uses an HIV-based lentiviral gene transfer vector^ref
• HHV-5 / CMV
• fomivirsen / ISIS 2922 (Vitravene^®; source : Isis Pharmaceuticals, Inc.) : antisense phosphorothioate oligoribonucleotide launched in 1998, but the success of protease inhibitors meant the market for the drug was smaller than anticipated
• HPVs
• afovirsen : antisense phosphorothioate oligoribonucleotide
• ZYC101a / ZYC101 / ZYC00101 / BIOTOPE-CD (source : Zycos) is a bacterial expression plasmid encoding a 13 amino acid sequence that is highly homologous with human papillomavirus E7 within poly (lactide-co-glycolide) microparticles (ZYC101) in women with HLA A2⁺ antigen and persistent cervical intraepithelial neoplasia grade 2/3 and HPV16. It is designed to specifically eliminate cervical high-grade intraepithelial lesion (HGIEL : CIN2/3) and anal dysplasia
• RNAi : anyway some escape mutants, with silent point mutations in the targeted sequence can emerge after treatment with RNAi. Thus, it may be necessary to attack viruses with multiple RNAi sequences simultaneously to prevent the emergence of resistant strains.
• cancer(in Europe over 80% of trials, while in the USA the figure is 72%)
• for lymphoproliferative disorders^ref
• immuno-gene therapy (29%)
• Leuvectin^® is a plasmid DNA/lipid complex comprised of a plasmid DNA expression vector (VCL-1102, 30) encoding human IL-2 complexed in a 5:1 mass ratio with DMRIE/DOPE lipid that has been developed for the treatment of cancer. DMRIE/DOPE is a cationic lipid, which facilitates in vitro and in vivo transfection of plasmid DNA. In vitro transfection with the IL-2 plasmid DNA/DMRIE/DOPE complex results in the expression of sustained levels of biologically active IL-2. Human tumor cell lines and primary human tumor cells established from biopsies were readily transfected in vitro resulting in the expression of IL-2. Following in vitro transfection, IL-2 expression continued up to several weeks post-transfection in primary tumor cells. In preclinical efficacy studies in a murine model of renal cell carcinoma (RCC), the direct intratumoral administration of an IL-2 plasmid DNA/DMRIE/DOPE complex resulted in the generation of tumor specific lymphocytes and complete tumor regression in the majority of the mice. In preclinical animal safety studies, repeated administration of Leuvectin was safe and well-tolerated. Following these promising preclinical results, Leuvectin has entered clinical trials and 2 pilot phase I/II trials are described^ref
• GDEPT (10%)
• CYP2B6 (MetXia^®; source : Oxford BioMedica plc) delivered via a third generation retroviral vector enables local production of the anti-tumour, cytotoxic derivative of the prodrug cyclophosphamide. Indications include all solid tumours where CPA may be used as chemotherapy. This includes breast, head and neck, ovarian, liver and pancreatic cancers^ref
• chemoprotection of stem cells by insertion of drug resistance gene(s) (5%)
• insertion of tumour suppressor genes (4%) : According to data published by the Journal of Gene Medicine, there were a total of 1020 approved gene therapy clinical trials in the world at the end of January 2005. Among these clinical trials, 66% were for the treatment of cancer. Of these cancer gene therapy trials, 58 used recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53). > 20 kinds of cancer indications have been treated with rAd-p53 agent, such as head and neck squamous cell carcinoma (HNSCC), lung cancer, breast cancer, liver cancers. Various clinical treatment regimens have been evaluated, including administration of rAd-p53 agent alone or in combination with conventional therapies such as radiotherapy, chemotherapy, and surgery.
• rAd.p53 / rAd/p53 is a pIX-deleted, wild-type p53 gene delivery system that is designed to lower the replication-competent adenovirus (RCA) occurrence
• p53 Ad / ACN53 / SCH58500 : a replication-deficient, type 5 adenovirus vector expressing human wildtype p53 tumor suppressor under the control of the CMV promoter
• head and neck squamous cell carcinoma (HNSCC) : injectable adenoviral vector to deliver p53 tumor suppressor genes at the precise site of cancer (Gendicine^®; source : Shenzhen SiBiono GeneTech Co., Ltd). After 8 weeks of therapy involving 1 injection per week on patients with late-stage disease, 64% of patients' tumors went into complete regression and 32% experienced partial regression. In combination with chemotherapy and radiotherapy, it improved treatment efficacy more than 3-fold. Over > 3 years, no patient relapsed. The only side effect was fever in around a third of patients^ref
• transfer of proapoptotic genes
• R2Fas : artificial VEGF receptor in which the intracellular region of VEGFR2 was replaced with a part of the Fas death receptor, which can trigger apoptosis
• LY2181308 is an anti-sense oligonucleotide that potently downregulated survivin expression in human cancer cells derived from lung, colon, breast, prostate, ovary, cervix, skin and brain
• inhibition of oncogenes
• ISIS 2503 (source : Isis Pharmaceuticals, Inc.) antisense inhibitor of H-RAS in metastatic pancreatic cancer
• ISIS 3521 / CGP 64128A (source : Isis Pharmaceuticals, Inc.) antisense inhibitor of PK-Ca for ovarian carcinoma
• MYCN antisense decrease tumorigenesis in a mouse model of neuroblastoma^ref
• inhibition of neovascularization
• chemosensitization :
• inhibition of bcl-2 expression by antisense oligodeoxynucleotides (ODN) might render bcl-2 overexpressing malignant B cells more susceptible to chemotherapy :
• NOV 2009 ODN does not contain CG dinucleotides
• G3139 / oblimersen sodium / augmerosen (Genasense^®; source : Aventis and Genta, Inc.), an 18-mer phosphorothioate antisense ODN corresponding to the first 6 exons of Bcl-2 mRNA is being investigated for use in blood, skin, colon, breast, lung, lymph and prostate cancer. It contains unmethylated CG dinucleotides (CpG) are known to activate B cells via TLR9
Without cationic lipids, no antisense-mediated inhibition of bcl-2 synthesis in malignant B cell lines and primary B-CLL cells was achieved with G3139 and NOV 2009. Instead, G3139, but not NOV 2009, induced similar changes as ODN 2006 in proliferation, expression of costimulatory and antigen-presenting molecules, as well as in bcl-2 and bcl-xL levels of primary B-CLL cells. G3139 and ODN 2006 inhibited in vitro, spontaneous apoptosis in B-CLL cells of patients with high serum thymidine kinase activity (s-TK, marker for proliferative activity of malignant B cells), whereas in patients with low s-TK activity, apoptosis was induced. In conclusion, results suggest that modulation of malignant B cell apoptosis by G3139 depends on its immunostimulatory properties rather than on antisense-mediated reduction of bcl-2 expression. Immunostimulatory CpG ODN may have a therapeutic potential in patients with B-CLL, especially those with low s-TK activity^ref.
• intradermal injection of plasmid DNA encoding a transcriptionally targeted cytotoxic gene, along with hsp70, not only promoted tissue-specific, inflammatory killing of normal melanocytes, but also induced a CD8⁺ T-cell–dependent, antigen-specific response in mice that eradicated systemically (metastatic) established B16 tumors. This CD8⁺ T cell response was subsequently suppressed in vivo within a few days. The data demonstrate that deliberate destruction of normal tissue can be exploited to generate immunity against a malignant disease originating from that tissue. This approach obviates the need to identify tumor antigens and does not require complex isolation of tumor cells or their derivatives. In addition, it provides a model system for studying the mechanisms underlying the etiology and control of autoimmune diseases. Finally, despite targeting normal tissue, therapy could be separated from development of overt autoimmune symptoms, suggesting that the strategy may be valuable against tumors derived from both non-essential and essential tissue types^ref. The researchers will pursue 2 basic paths. One will extend the current work on a heat shock vaccine to other tissue and tumor types to determine its effectiveness against breast, lung or prostate cancers. The other is to test this immunotherapy in clinical trials with humans.
• safety switches for T lymphocytes :
• immunogenic bacterial or viral suicide genes :
• HSV thymidine kinase (HSV-TK) - hygromycin phosphotransferase (Hy) fusion gene can be selected on the basis of hygromycin resistance and then eradicated if adverse effects occur by administration of gangiclovir which will be converted by Tk to a cytotoxic agent. However, the viral-derived Tk product and bacterial-derived Hy products proved immunogenic presenting multiple peptides that could generate a cytotoxic T cell response that effectively eradicated transduced cells. Since multiple epitopes could be recognized, modification to remove immunogenic sequences will not be possible, and as the Tk-Hy–specific immune response contains memory cells, it will persist long term^ref
• HSV thymidine kinase (HSV-TK) and nerve growth factor receptor (NGFR) : a retroviral vector (SFCMM-2)^ref for the transfer and expression into human PBLs of 2 genes: The first encodes a truncated (nonfunctional) form of the human low-affinity receptor for nerve growth factor (DLNGFR); the second encodes the HSV-TK-NEO fusion protein. DLNGFR is located on the cell surface and allows rapid in vitro selection of transduced cells by the use of magnetic immunobeads. In addition, a surface marker allows easy ex vivo detection and characterization of the transduced cells by FACS analysis^ref
• nonimmunogenic human suicide genes :
• chemical inducers of dimerization (CIDs) and endogenous proapoptotic molecules : human apoptosis molecules can be linked to FK506 binding proteins that contain a binding site for a CID. Administration of this drug then results in the formation of a complex of 2 apoptosis-fusion molecules, which leads to their activation and thus apoptosis.
• Fas^ref
• caspase 9^ref : may kill a higher percentage of transduced cells, including those that have unregulated inhibitors of the apoptosis pathway
Tumor-specific promoterscan be used. Whether tumour progression can be reversed by inhibition of a single oncogene is an important question that is under debate. Following regression, most patients develop tumours that have progressed to a oncogene-independent state.
Web resources : International Society for Cancer Gene Therapy (ISCGT)
• cholestasis-induced liver fibrosis. Transfected gene : SOD2 and SOD3, which block formation of ROS and production of toxic cytokines thereby minimizing liver injury caused by cholestasis.
• anemia : erythropoietin viral construct under the control of HRE (Repoxygen^®; source : Oxford BioMedica plc) for i.m. injection. Athletes may consider using Repoxygen as a means of increasing RBC mass. However in principle, if Repoxygen acts as described, it should not raise RBC levels above normal due to it's self-regulating properties and therefore may be a poor choice for doping purposes^ref. An estimated US$2 billion is spent annually on recombinant Epo in the US dialysis market while oncology-related anaemia is an estimated US$3 billion market. Delivery of EPO into cynomolgus macaques through intramuscularly administered AAV vectors. As expected, the animals developed supraphysiologic levels of EPO and polycythemia. However, severe anemia ensued in some animals because of an autoimmune response to endogenous and transgene derived EPO. This is the first example of gene therapy leading to inadvertent auto-immunity in primates^ref
• bone regeneration

Transfected genes :
• BMP2
• BMP4
• BMP7
• cardiovascular gene therapy
• inhibition of neovascularization
• ... of eye (after trauma or in proliferative vitreoretinopathy (PVR)), which often results in visual loss: conventional treatment for these disorders are based on laser photocoagulation and cryotherapy, which are themselves inherently destructive, have significantly potential to adversely affect vision, and whose effects are rarely sustained as they don't control the pro-angiogenic mechanisms arising from the underlying disease.

Transfected genes :
• soluble Flt1 / VEGFR1
• pegaptanib (Macugen^®; source : Eyetech Pharmaceuticals, Pfizer) is a pegylated aptamer that binds to VEGF-A₁₆₅ impairing binding to VEGFR^ref
• RetinoStat^® (source : Oxford BioMedica plc) consists of a LentiVector system delivering a gene and encoding an angiostatic protein, endostatin and angiostatin, under the control of the HRE element for the treatment of vision-loss in age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR) via localised gene delivery^ref
• soluble TGF-btype II receptor
• ... of tumors (tumour angioneogenesis). Transfected genes :
• anti-laminin scFv
• serpin antithrombin
• uPAR competitive inhibitors (e.g. uPA amino-terminal fragment (uPA_1-135 or ATF)) : this prevents plasminogen conversion into plasmin, and subsequent activation of the proteolytic cascade required for ECM degradation and cell invasion. The uPA binding to its specific receptor uPAR greatly potentiates plasminogen activation on the cell surface. Generated plasmin is a wide range serine protease which directly degrades some of the components of the ECM such as fibronectin or laminin, but can also promote local degradation of the stroma by its ability to convert inactive zymogens into active MMPs.
• soluble Flt1 / VEGFR1
• IFN-a₁
• IFN-b
• IL-1ra
• CXCL4 / platelet factor 4 (PF4)
• RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5' UTR of early growth response (EGR-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices
• chimeric death receptor composed of elements of CD178 / FASL and TNFR1 under the control of the endothelial-cell-specific pre-proendothelin-1 (PPE-1) promoter (a vasoconstrictor and smooth muscle cell mitogen synthesized by endothelial cells whose gene promoter contains a hypoxia-responsive element (HRE) that increases gene expression only under hypoxic conditions, such as in the tumour microenvironment) modified to contain 3 HREs, designed to activate the FAS-induced apoptotic pathway following binding to TNF-a, which is abundant in the tumour microenvironment. When delivered by AAV, it stays on target and does not elicit immune responses. Expression of the chimeric receptor did not cause liver damage in mice, which was a concern because FASL administration has been previously shown to destroy hepatocytes^ref
• endogenous inhibitors of angiogenesis
• induction of neoangiogenesis and vasculogenesis to ...
• overcome myocardium or peripheral ischemia
• accelerate healing of wound that are otherwise difficult to treat, such as those in patients with metabolic disorders or infections
• prevent bronchopulmonary dysplasia (BPD) in premature babies (hyperoxia-induced lung injury)^ref
Transfected genes :
• VEGFA (₁₂₁, ₍₁₄₅₎, ₁₆₅, ₍₁₈₃₎, ₁₈₉ or ₂₀₆) (VIVA trial) : AAV delivering VEGF₁₆₅ improves survival of ischemic cutaneous and musculocutaneous flaps^ref. The prolonged expression of VEGF in the normoperfused skeletal muscles of adult rodents after gene transfer using AAV vectors induces the formation of a large set of new capillaries and small arteries. Notably, this process is accompanied by the massive infiltration by mononuclear cells. This observation raises the possibility that these cells might represent circulating progenitors that are eventually incorporated in the newly formed vessels. In 4 different experimental models [based on: a) the transplantation of male bone marrow into female recipients; b) the transplantation of Tie2-GFP transgenic bone marrow; c) the transplantation of bone marrow in the presence of erythropoietin (EPO), a mobilizer of endothelial progenitor cells (EPCs); d) the re-implantation of ex vivo expanded EPCs], VEGF acted as a powerful attractor of bone marrow-derived mononuclear cells, bearing different myeloid and endothelial markers proper of EPCs, to the sites of neovascularization. In no case, however, the attracted cells were incorporated in the newly formed vasculature. These observations indicate that new blood vessel formation induced by VEGF occurs through bona fide sprouting angiogenesis; the contribution of the infiltrating bone marrow-derived cells to this process still remains enigmatic^ref. VEGF₁₆₅ gene delivery via AAV exerts a marked beneficial action by enhancing both arteriologenesis and cardiomyocyte viability in infarcted myocardium^ref. rAAV-mediated gene delivery of VEGF₁₆₅ into cardiac muscle, during acute myocardial infarction, exerts a protective effect to promote long-term functional recovery. Acute infarction of the anterior LV wall was induced in 12 chronically instrumented dogs by permanent occlusion of the LAD coronary artery. Four hours after occlusion, rAAV-VEGF₁₆₅ or rAAV-LacZ (n=6 each; 5x10(12) viral particles per animal) was directly injected with an echo-guided needle into the dysfunctional cardiac wall. LV and arterial pressure, dP/dtmax, and ejection fraction were not significantly different between the two groups over time. In contrast, in the infarcted region, at four weeks after infarction, fractional shortening was 75+/-18% and -3+/-15% of baseline and length-pressure area was 54+/-15% and 0.8+/-15% of baseline in VEGF₁₆₅ versus LacZ, respectively (P<0.05). Histological analysis of the border regions showed a marked increase in the number of a-SMA-positive arterioles (68+/-2.8 versus 100+/-3.8 vessels per microscopic field in LacZ and VEGF₁₆₅, respectively; P<0.05). In both groups, VEGFR-2 was diffusely expressed on the surviving cardiomyocytes and, consistently, myocardial viability was significantly improved in the VEGF₁₆₅-treated group, with several troponin T-expressing cardiomyocytes displaying nuclear positivity for the proliferation marker PCNA. Altogether, our results indicate that VEGF₁₆₅ gene delivery exerts a marked beneficial action by enhancing both arteriologenesis and cardiomyocyte viability in infarcted myocardium^ref.
• VEGFB^ref
• VEGFC
• VEGFD
• PlGF
• FGF-1 / aFGF^ref
• FGF-2 / bFGF (TRAFFIC trial)
• FGF-4(AGENT trial)
• FGF-5
• HGF / SF
• Ang-1
• Ang-2
• PDGF bb
• HIF-1a (it is expecially useful as it is a TF for many of the above mentioned genes)
• GH (Biobypass by GenVec Inc.)
Transgene expression must be strictly time-regulated. Hypoxia represents an endogenous pathophysiological signal underlying cell growth, adaptation and death in a variety of diseases, including ischemic heart diseases, stroke and solid tumors. A vigilant vector system depends on a gene switch which can sense the hypoxia signal occurring in ischemic events and turn on/off protective gene expressions when necessary. This system uses the oxygen-dependent degradation domain derived from HIF-1a as the hypoxia sensor and a double-vector system as signal amplifier. For treating ischemic heart diseases, a cardiac-specific MLC-2v promoter is used to deliver transgenes specifically to the heart. When tested in cardiomyocyte cultures, it produced a rapid and robust gene induction upon exposure to low oxygen. In a mouse model for myocardial infarction, the vigilant vectors turned on therapeutic genes such as heme oxygenase-1 in response to ischemia, significantly reduced apoptosis in the infarct area and improved cardiac functions. The hypoxia-regulated gene transfer afforded by the vigilant vectors may provide a powerful tool for delivering therapeutic proteins specifically to ischemic tissues with optimal physiological control^ref.
• wound healing

Transfected genes :
• genes for induction of neoangiogenesis
• FGF-7 / KGF
• transient cutaneous adenoviral delivery of the host defense peptide hCAP-18/LL-37 is significantly more effective than administration of synthetic host defense peptides and might be a potential adjunct for wound treatment in the near future^ref
• cardiopathies : in vivo transcoronary gene delivery to the adult myocardium using immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic catheter-based segmental injection of cardioplegic solution containing substance P and recombinant Ad and AAV vectors
• (congestive) heart failure (CHF) : transfected genes :
• b₁-AR
• V₂
• phospholamban^{ref1, ref2, ref3}
• SERCA2a^{ref1, ref2, ref3, ref4, ref5}
• Bcl-2
• b-ARKct, a peptide which inhibits bARK1^{ref1, ref2}
• GTP binding protein overexpressed in skeletal muscle (GEM)^ref
• transplantation of MSCs overexpressing the Akt transgene to prevent hypoxic necrosis^ref
• when protected by AT2R through cardiac transduction via lentiviral vectors, LVWT was 85% lower, HW/BW was 91% and myocardial fibrosis was 43% lower after a two-week infusion of angiotensin II (AT2), which induced hypertension. Only 30-40% of cardiac cells was transduced and it appears that this amount is sufficient. while the AT2R prevented hypertrophy and fibrosis of the heart, the perivascular fibrosis (around peripheral blood vessels) was not reduced, nor was blood pressure : in heart failure, you may or not have high blood pressure present, so this is very good: to be able to affect the heart, without affecting the vasculature
• creation of pacemaker cells in myocardium. Transfected gene : inhibitor of K⁺ ATPase.
• prevention of post-percutaneous translumenal coronary angioplasty (PTCA) restenosis, in-stent restenosis and by-pass graft stenosis  : the aim is to prevent proliferation and migration of smooth muscle cells and consequent neointimal hyperplasia. Various types of catheters are availbale for gene tranfer into the vessel wall. Unfortunately, even with powerful viral vectors gene transfer efficiency through human atherosclerotic lesions and lipid-rich atheroma is < 5%, and results in a potentially harmful biodistribution of the vector. Therapeutic ultrasound has also proven efficient in augmenting intravascular gene delivery. For the treatment of in-stent restenosis a good method to deliver vectors locally to the target area is the stent itself. Transfected genes / targets :
• post-PTCA restenosis / in-stent restenosis
• intimal hyperplasia
• VEGFA
• VEGF-C
• VEGF-D
• NF-kB and E2F decoys
• TIMP-1
• c-myc antisense
• Gax
• p27-p16 fusion gene
• Ras
• p21
• TP53
• CD178 / FasL
• uteroglobin
• blocking PDGF or TGF-b
• PDGFbR antisense
• DN-RhoK
• iNOS / NOS2
• eNOS / NOS3
• TK
• ATF.BPTI
• HGF
• EC-SOD / SOD1
• DN-ERK
• DN-JNK
• re-endothelization
• VEGFA
• BioBypass-CAD^®
• BioBypass-PAD^®
• VEGF-C
• VEGF-D
• HGF
• thrombosis
• TFPI / LACI
• vein graft stenosis
• intimal hyperplasia
• TIMP-3
• E2F decoy dsODN
• reendothelization
• CNP
• u-PA
• arterial hypertension
• mutant angiotensinogen
• AT_1a receptor antisense
• ACE antisense
• hypercholesterolemia : researchers from the biotechnology company Alnylam, based in Kulmbach, Germany, and Cambridge, Massachusetts, showed in November 2004 that siRNAs can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs (by attaching a cholesterol molecule to it) resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced LDL-Ch by 44% and total cholesterol. Cells that make cholesterol have cholesterol receptors on their surface, so that they can sense levels of the molecule in the blood. Adding the cholesterol molecule to the RNA ensures that the complex is taken up specifically by these cells, in the liver and small intestine. These siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site^ref. The researchers checked the activity of a few genes unrelated to their target gene, and found that they were not affected. The doses used in the mice were quite high, and would be costly and impractical to give to people. And it's not clear how often a person would need to be injected with the treatment, or whether it could be delivered in some other form
• obesity :
• leptin gene therapy
• central : stereotactic hypothalamic delivery under the control of a time-specific promoter : this is required as :
• central overexpression of leptin (coupled with a precipitous fall in serum leptin) compromises the immune and reproductive systems and decreases bone mass.
• peripheral overexpression of leptin leads to pancreatic b cell disfunction and up-regulates SOCS3, which may interfere with insulin signaling.
• peripheral : electroporational transfer of naked plasmid with leptin cDNA (attached to the human insulin precursor secretion signal peptide gene) into skeletal muscle of both young and adult female mice. During the time of exogenic gene expression, daily food intake of leptin cDNA-treated mice was observed to be lower than the control. The body weight gain was prevented efficaciously regardless of if they were young or adult. At the 7th week after gene transfer, the body weight of both young and adult leptin cDNA-treated mice was about 20% lighter than the control. Although the body weight of pair fed controlled adult mice was close to the leptin cDNA-treated mice at the 8th week, they were always heavier than the leptin cDNA-treated mice before this time. The levels of retroperitoneal fats and serum TG of leptin cDNA-treated mice were markedly lower than that of the control. The relative serum hyperleptinemic level could last for about 2 months. The expression of leptin cDNA in muscle cells was also detected by RT-PCR. The levels of serum insulin and glucose of leptin cDNA-treated mice decreased slightly. Hyperleptinemia resulted in uterus expansion in young mice, but not in the adults^ref
• neurological diseases : gene products may be imported into the host CNS via stereotaxic injection of viral vectors for gene therapy, or autograft of genetically modified non-neural cells that can reside within the CNS to deliver exogenous factors to neurons : however, this approach is limited to correction of disease only in the vicinity of the graft and they lack the ability to incorporate widely into host cytoarchitecture in a functional manner following implantation, preventing reforming of essential circuits. Anyway the best choice for widely disseminated neurological disorders is NSCs transplantation.
• analgesia

Transfected genes :
• POMC
• IL-10 epidurally
• increasing GABA in rostral agranular insular cortex (RAIC) produces lasting analgesia by enhancing the descending inhibition of spinal nociceptive neurons^ref (both the anterior cingulate cortex and insular cortex receive pain messages and are thought to process motor and emotional responses to pain)
• glutamic acid decarboxylase (GAD) delivered with HHV into the spinal cord to increase production of GABA in a sciatica rat model. After injection under the skin, the virus did what it does best: it entered peripheral nerves and travelled up them to the nerve centres. A normal herpes virus would then replicate and travel back down the nerve to the skin where it first entered, causing sores. But the crippled version used by the scientists stayed put in nerve centres and expressed the GAD gene, raising local levels of GABA^ref. The treated rats became significantly less sensitive to touch and warmth, whereas rats that underwent a sham treatment did not. The effect lasted up to 6 weeks: the lifespan of the particular gene promoter that the scientists used. Fink hopes to start clinical trials of the therapy in humans in 2006
• Alzheimer's disease (AD) :
• neprilysin / neutral endopeptidase / enkephalinase / CD10 / CALLA
• NGF stimulates cholinergic function (cholinergic neuron loss is a cardinal feature of AD), improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. A phase 1 trial of ex vivo NGF gene delivery in 8 individuals with mild AD, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in 6 subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and AD Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-FDG after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for AD are warranted^ref.
• humanin (source : Neurologix, Inc.)
• Canavan disease : stereotaxic injections of AAV encoding aspartoacylase
• Parkinson’s disease (PD)

Transfected genes :
• DOPA-decarboxylase
• GAD1 in AAV
• Tet-on GDNF in rAAV3
• ProSavin^® (source : Oxford BioMedica plc) consists of a LentiVector carrying 3 genes encoding the enzymes needed for dopamine synthesis for the treatment of late stage PD^ref
• dominant progressive brain disorder
• spinocerebellar ataxia type 1 (SCA1) : RNA interference via AAV in mice
• Huntington's disease (HD):

Transfected genes :
• CNTF
• siRNA via HIV-1 targeting a SNP that appears in 70% of mutated IT15 / huntingtin alleles (not targeting the expanded trinucleotide itself). Completely silencing the gene in people with the disease is not an option because neurons may not survive without the protein. RNA interference directed against mutant human htt reduced htt mRNA and protein expression in cell culture and in HD mouse brain. Importantly, htt gene silencing improved behavioral and neuropathological abnormalities associated with HD^ref. It has also been used in human cells in culture dishes
• anti-N-terminal huntingtin intrabodies (C4 sFv) reduce aggregation and cellular toxicity in vitro and in a Drosophila model of HD. In the presence of the C4 sFv intrabody, the proportion of HD flies surviving to adulthood increases from 23% to 100%, and the mean and maximum lifespan of adult HD flies is significantly prolonged. Neurodegeneration and formation of visible huntingtin aggregates are slowed^ref.
• sensory hearing loss : ATOH1 / Math1, a key regulator of auditory hair cell development, into non-sensory epithelial cells that remain in the deafened inner ears of adult guinea pigs, whose original hair cells were destroyed by exposure to ototoxic drugs
• amyotrophic lateral sclerosis (ALS) / Lou Gehrig's disease : MoNuDin^® (source : Oxford BioMedica plc) consists of a LentiVector delivering the VEGFA slowed the development of the illness and increased the life expectancy of the affected mice by 30%, with no toxic side-effects^ref
• temporal lobe epilepsy : galaninsecretion gene therapy (SGT) in neurons via AAV vectors
• glaucoma : BDNF, Bcl-2and FGF-2 / bFGF in ganglion cells
• retinitis pigmentosa
• autosomal recessive : rod photoreceptor-specific cGMP PDE
• autosomal dominant : in photoreceptor cells
• spinal cord injuries (SCI): Innurex^® (source : Oxford BioMedica plc) consists of a LentiVector delivering the human RARß2 gene, a proprietary nuclear retinoic acid receptor gene, to induce neurite outgrowth in damaged nerves to repair spinal cord injury^ref
• most degenerative (traumatic or toxic) neuropathies of PNS : NT-3, BDNF
• age-related macular degenerations (ARMD) : Bcl-2 , BDNF, NGF, NT-3, CNTF, FGF-2 / bFGF; Alnylam will bring its first “RNAi therapeutic” into the clinic in 2005 to target VEGFA
• drug addictions
• cocaine addiction : bacteriophage have the capacity to penetrate the CNS when administered intranasally. A method is presented for engineering filamentous bacteriophage to display cocaine-binding proteins on its surface that sequester cocaine in the brain. The virus lingers in the brain for around two weeks, so although they might relapse once, the absence of any euphoric feeling would then discourage them from taking it again^ref
• ethanol addiction : D₂ receptor
• inflammation
• IL-1RA
• IL-10
• autoimmune diseases :
• immunosuppression
• antigen-specific immunosuppression
• TcR mimic peptide (TCRpep : an 8-amino-acid peptide, homologous to the transmembrane region of the TcR a chain) secretion gene therapy (SGT) in dendritic cells (DCs) induces antigen specific immunosuppression, because transduced DCs that did not express the respective antigen failed to convey protection in allergy and EAE models
• generalized immunosuppression
• acute and chronic liver injury : i.v. siRNA-directed Fas silencing.
• inflammatory bowel diseases (IBDs) : CD54 / ICAM-1 antisense oligonucleotide (ISIS 2302) prevents local recruitment of WBCs
• type I hypersensitivities : topically applied NF-kB decoy ODN.
• transplant rejection : ER-directed anti-human MHC I scFv intrabody directed against a monomorphic, conformational epitope, expressed across species lines, on the MHC I heavy chain.
• rheumatoid arthritis: intraarticular injection of replication-defective MoMLV or AAV encoding IL-1Ra
• type I diabetes mellitus : mature insulin has to be
• expressed from a site distinct from pancreatic b-cells
• transfection of hepatocytes with insulin expression cassette
• transfection of hepatocytes with the carboxyl terminus of PDX1 / IPF1 fused to the VP16 activation domain from HSV, under the control of transthyretin promoter. Xenopus laevis tadpoles containing the TTR-Xlhbox8-VP16:elastase-GFP transgene has been shown to form both exocrine and endocrine pancreas in an area normally occupied by liver thanks to transdifferentiation rather than change of a developmental pathway in the embryo. Once established, the ectopic pancreas persists although TTR expression is downregulated as the conversion to pancreas occurs (hence not requiring permanent genetic change). Mice treated with HDAD-Ipf1 developed fulminant hepatitis, however, because of the exocrine-differentiating activity of Ipf1. HDAD-mediated transfer of NeuroD1 (a factor downstream of Ipf1) and betacellulin (Btc) (a growth factor), doesn't produce hepatitis.
• expression has to be regulated appropriately. The most efficient production of mature insulin is obtained by exploiting endogenous furin to process an appropriately modified pro-insulin, although co-expressing PC2 and PC3 does enhances the production of mature insulin from wild-type pro-insulin. Although hepatocytes and intestinal K cells express GLUT-2, GK and a K_ATP channel similar to that expressed in pancreas, they are not able to provide the desired physiologic regulation of the gene in response to blood glucose. Time-specific regulation of insulin expression can theoretically be achieved  ...
• at the transcriptional level (time-specific transcriptional complexes) :
• physiologic cues (ie glucose-induced) : glucose-sensitive promoters
• pharmacological cues
Exogenous insulin expression from the vector is induced several hours after the hyperglycemic peak, because of the delay due to transcription and translation of the insulin product. A similar interval of time is required to reverse hormone expression to negligible levels upon return to a normoglycemic status, due to the requirement to suppress transcription that is mediated by inducible transcription factors and to the half-life of the mRNA. The net result is an induction of insulin that is delayed beyond the peak of blood glucose and persists during post-prandial episodes of hyperglycemia. In humans, metabolic control using this approach would not be feasible exposing patients to dangerous fluctuations. Nevertheless, pharmacological (but not glucose-) regulated transcription may be a feasible way to obtain therapeutic baseline levels of the hormone in a gene therapy setting : in many ways transcriptional regulation can replace and potentially improve upon the requirement of long acting insulin. This, however, would have to be supplemented by administration of short acting insulin or by pharmacologically regulated secretion of recombinant insulin already synthesized and stored in subcellular compartments.
Attempts at obtaining physiologic control of insulin secretion either using cells equipped with the ability to store the recombinant hormone and release it upon a glucose-stimulus or converting hepatocytes into b cells, are promising but preliminary.
• at the exocytosis level
• pharmacological cues
• graft tolerance : chronic renal allograft rejection can be prevented effectively by local delivery of recombinant adeno-associated virus (AAV) vectors that encode the CTLA4Ig immunosuppressant protein to the donor kidney in a fully MHC-mismatched rat strain combination. AAV CTLA4Ig prevented progressive proteinuria and protected transplant kidneys from renal structural injury. A population of anergic T cells with regulatory activity, which eventually were responsible for the induction of tolerance, were found in recipient lymph nodes and in the graft as long as 120 d after transplantation. These data indicate that AAV-mediated CTLA4Ig gene transfer to donor graft represents a promising tool to prevent the onset of chronic rejection and circumvent the unwanted systemic adverse effects of the administration of immunomodulatory protein^ref

• Associations :
• American Society of Gene Therapy (ASGT)
• European Society of Gene Therapy (ESGT)
• Oligonucleotide Therapeutics Society (OTS)
• Centre for Gene Therapy (CGT) of Childhood Disease
• Center for Cell and Gene Therapy - Baylor College of Medicine
• UK Gene Therapy Advisory Committee (GTAC)
• Gene Therapy Initiative: Harvard Institute of Human Genetics
• Institute for Human Gene Therapy (IHGT)
• Powell Gene Therapy Center at the University of Florida
• Department of Therapeutic Gene Modulation
• NIH's pilot Human Gene Transfer database
• since 1999, the hospital San Raffaele HSR Human Gene Therapy Program was split into an academic (Telethon Institute of Gene Therapy (TIGET) and the Cancer Gene Therapy and Immunotherapy Program (CGTIP)) and an industrial (MolMed S.p.A.) component.
• Journals :
• Gene Therapy (impact factor 2003 : 5.293)
• Genetic Vaccines and Therapy (impact factor 2003 : ?)
• Human Gene Therapy (impact factor 2003 : 4.965)
• Molecular Therapy (impact factor 2003 : 6.125)
• The Journal of Gene Medicine (impact factor 2003 : 4.567)
• researchers in the field of gene therapy :
• Mauro Giacca (ICGEB Molecular Medicine Group, Trieste)
• Vincenzo Bronte (Istituto Oncologico Veneto - UOA Immunologia e Diagnostica Molecolare e Oncologica)

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