Gene Therapy Laboratory

Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation. This block is caused by mutations of the gene encoding the gammac cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors. After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective gammac Moloney retrovirus-derived vector and ex vivo infection of CD34+ cells. After a 10-month follow-up period, gammac transgene-expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit.

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10(11) and 9.8 x 10(12)/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.

Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients. Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo. Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Française contre les Myopathies/Généthon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year. Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly. Our results indicate that: (i) rAAV yields ranged between 1011 to 5 × 1012 total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio. Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA. In summary, this study describes a general method to titrate rAAV, independently of the transgene and its expression, and to measure the level of contamination with adenovirus and rep-positive AAV. Furthermore, we report a new production procedure using adenoviral plasmids instead of virions and resulting in rAAV stocks with undetectable adenovirus contamination. Production of recombinant adeno-associated virus (rAAV) relies upon a complex and relatively inefficient purification procedure. Thus, a precise titration of the rAAV stock, as well as its characterization to measure contaminating adenovirus and rep-positive AAV particles, are essential to evaluate the efficiency of these vectors in vitro and in vivo. We describe here the development of a modified RCA using a stable rep-cap HeLa cell line. Eighteen large-scale rAAV stocks, produced by our vector core during the past year, were titered and characterized using this modified RCA. This assay was used to monitor the consequences of some major modifications introduced in the rAAV production procedure and particularly the use of an adenoviral plasmid to produce rAAV stocks free of infectious adenoviral particles.

Adeno-associated viruses (AAV) are small, nonenveloped single-stranded DNA viruses which require helper viruses to facilitate efficient replication. These recombinant viruses are some of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Nevertheless, the presence of humoral responses to the wild-type AAV common among humans is one of the limitations of in vivo transduction efficacy in humans using cognate recombinant vector. In this study, based on the serum samples that we were able to collect from various clinical situations, we studied the impact of one to five plasmapheresis (PP), at 1-5 day intervals on neutralizing factor (NAF) titers specific for AAV types 1, 2, 6, and 8 in seropositive patients with diverse pathologies and immunosuppressor treatments. We show that frequent sessions of PP result in drastic reduction of NAF specific for AAV1, 2, 6, and 8 to undetectable levels or titers <1:5, mainly when initial titers, i.e., before the first PP were ≤1:20. Altogether, these results show that the use of PP and its possible association with pharmacological immunosuppressive treatments may help to design optimal management of seropositive patients for AAV gene therapy treatments.

A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD.

We previously described chimeric recombinant adeno-associated virus (rAAV) vectors 2/4 and 2/5 as the most efficient vectors in rat retina. We now characterize these two vectors carrying the CMV.gfp genome following subretinal injection in the Wistar rat, beagle dog, and cynomolgus macaque. Both serotypes displayed stable GFP expression for the duration of the experiment (6 months) in all three animal models. Similar to the AAV-2 serotype, AAV-2/5 transduced both RPE and photoreceptor cells, with higher level of transduction in photoreceptors, whereas rAAV-2/4 transduction was unambiguously restricted to RPE cells. This unique specificity found conserved among all three species makes AAV-2/4-derived vectors attractive for retinal diseases originating in RPE such as Leber congenital amaurosis (RPE65) or retinitis pigmentosa due to a mutated mertk gene. To provide further important preclinical data, vector shedding was monitored by PCR in various biological fluids for 2 months post-rAAV administration. Following rAAV-2/4 and -5 subretinal delivery in dogs (n = 6) and in nonhuman primates (n = 2), vector genome was found in lacrymal and nasal fluids for up to 3-4 days and in the serum for up to 15-20 days. Overall, these findings will have a practical impact on the development of future gene therapy trials of retinal diseases.

There are more than 40 different forms of inherited lysosomal storage diseases (LSDs) known to occur in humans and the aggregate incidence has been estimated to approach 1 in 7000 live births. Most LSDs are associated with high morbidity and mortality and represent a significant burden on patients, their families, and health care providers. Except for symptomatic therapies, many LSDs remain untreatable, and gene therapy is among the only viable treatment options potentially available. Therapies for some LSDs do exist, or are under evaluation, including heterologous bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and substrate reduction therapy (SRT), but these treatment options are associated with significant concerns, including high morbidity and mortality (BMT), limited positive outcomes (BMT), incomplete response to therapy (BMT, ERT, and SRT), life-long therapy (ERT, SRT), and cost (BMT, ERT, SRT). Gene therapy represents a potential alternative therapy, albeit a therapy with its own attendant concerns. Animal models of LSDs play a critical role in evaluating the efficacy and safety of therapy for many of these conditions. Naturally occurring animal homologs of LSDs have been described in the mouse, rat, dog, cat, guinea pig, emu, quail, goat, cattle, sheep, and pig. In this review we discuss those animal models that have been used in gene therapy experiments and those with promise for future evaluations.

We reported previously that controlled expression of a foreign gene in response to tetracycline derivative can be accomplished in mice by the autologous transplantation of retrovirus-modified muscle cells. Although regulated systemic delivery of therapeutic proteins from engineered tissues has potential clinical application, the transplantation of muscle cells is not currently feasible in humans. Several studies have shown that a single injection of adeno-associated virus (AAV) vectors into mouse muscle results in long-term expression of reporter genes as well as sustained delivery of proteins into the serum. Because this method is potentially applicable clinically, we constructed an AAV vector in which the expression of the mouse erythropoietin (Epo) cDNA is modulated in response to doxycycline. The vector was injected intramuscularly in normal mice. We observed that hematocrit and serum Epo concentrations could be modulated over a 29-week period in response to the presence or absence of doxycycline in the drinking water of these animals. Thus, a regulated gene expression cassette can be incorporated into a single AAV vector, such that intramuscular injection of the vector allows sustained and regulated expression of a desired gene.

Previous studies on distribution and toxicity of viral vectors administered in monkeys indicated that the nonhuman primate model has a reasonable predictive value for clinical applications. In this study, eight macaques were injected intramuscularly with recombinant adeno-associated virus (rAAV) at doses similar to those administered to hemophilia B patients, and followed to analyze the dissemination and shedding in biological samples and long-term persistence in distant organs. Following rAAV delivery, we found vector genome in various biological fluids for up to 6 days and infectious particles exclusively in the serum during the first 48-72 hours. rAAV sequences were detected in peripheral blood mononuclear cells (PBMC) for up to 10 months. At necropsy, 8 to 18 months after rAAV delivery, rAAV sequences were found in lymph nodes and livers but never in the gonads. Tissue examination, of liver in particular, showed no abnormalities. We concluded that during our experimental time frame, rAAV-mediated gene transfer into skeletal muscle of macaques seemed to be safe with respect to the recipient and the environment. However, it was associated with a transient viremia and the persistence of rAAV sequences in PBMC, lymph nodes, and liver, the long-term consequences of which remain unknown.

Recombinant adeno-associated virus 2/5 (rAAV2/5), a hybrid rAAV-2 with AAV-5 capsid, seems to be a very efficient delivery vector for the transduction of the lung adenocarcinoma cell line A549. Infection of the A549 cell line with a rAAV2/5 vector encoding the extracellular domain of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, amino acids 114-281) resulted in secretion of soluble TRAIL (sTRAIL) and induction of apoptosis in these cells. rAAV2/5-sTRAIL mediated delivery and stable expression of sTRAIL resulted in the presence of the trimeric form of sTRAIL in sera of nude mice that were implanted with s.c. or orthotopic A549 tumors. The rAAV2/5-sTRAIL transduction of the tumors resulted in a statistically significant reduction in tumor growth and prolonged survival of the tumor-bearing animals. Primary cell culture, histologic examination of the tumors, and serum analyses showed the absence of detectable TRAIL-induced toxicity in normal tissues including the liver. The successful inhibition of lung cancer growth and the absence of detectable toxicity suggest a putative role for rAAV2/5-sTRAIL(114-281) in the therapy of lung cancer.

BACKGROUND: Recently, several reports have described the ability of recombinant baculoviruses to transduce a variety of mammalian cells. Yet, mechanisms involved in baculovirus entry in those cells remain largely unexplored, particularly at the primary binding step of the virions to the cell membrane. METHODS: This report focused on the primary virus-cell interactions that lead to in vitro transduction of human 293 cells using a polyhedrin-deleted baculovirus harboring a CMV-driven beta-galactosidase gene (BacLacZ). RESULTS: Infection rate monitored for 8 h and transduction rate with a multiplicity of infection of up to 800 were, both, non-saturable. Temperatures from 37 degrees C to 4 degrees C dramatically impaired BacLacZ but not adenovirus cell attachment. Competitive infections performed with an excess of a non LacZ-expressing baculovirus hardly competed at a 1/1 ratio. Consistent with an adsorptive binding process onto the cell surface, interactions through electrostatic charges between both viral and cell membranes appeared to be critical for BacLacZ transduction. The addition of polybrene to the cells prior to or during the infection prevented both virus binding and LacZ gene transfer, suggesting the involvement of negatively charged epitopes exposed at the cell surface. The simultaneous presence of the highly charged heparin abrogated BacLacZ binding to the cell surface and subsequent gene transfer. Lastly, direct in vitro binding of BacLacZ to heparin but not BSA columns could be demonstrated after elution of infectious BacLacZ virus in high salt molarity. CONCLUSION: Electrostatic charges play a critical role during the first step in mammalian cell transduction mediated by a recombinant baculovirus.

BACKGROUND: A possible procedure for the production of clinical grade recombinant adeno-associated virus type 2 (rAAV) would include the use of packaging cell lines, harboring the rep-cap genes and the vector, combined with a replication defective adenoviral plasmid to provide the helper activities. Several studies have already shown that rAAV can be efficiently assembled by infecting the stable packaging cell line with adenovirus. However, the direct comparison with an adenoviral plasmid has never been reported. METHODS: To investigate this point, a clone of HeLa and 293 cells harboring one to two rep-cap copies per cell genome (HeRC32 and 293RC21, respectively) were generated. Recombinant AAV was produced by transiently transfecting the AAVCMVLacZ vector and supplying the adenoviral helper activities by either wild-type adenovirus or an adenoviral plasmid (pAdc). As a control, rAAV was similarly produced from naive Hela and 293 cells additionally transfected with a rep-cap plasmid. RESULTS: Despite satisfactory rAAV yields from Hela and 293 cells, we show that those from HeRC32 and 293RC21 cells dramatically decrease when adenovirus is replaced by the adenoviral plasmid (pAdc). The analysis performed to identify the factors hampering efficient rAAV assembly by HeRC32 cells in the presence of pAdc shows that: (1) while upon adenovirus infection the integrated rep-cap genome undergoes a dramatic amplification leading to a 100-fold increase in the rep-cap copy number, no amplification is detected upon transfection of pAdc; (2) in pAdc-transfected HeRC32 cells, the intracellular localization of the adenovirus E4orf6 and E1B-55kDa proteins is abnormal as compared to adenovirus-infected cells. CONCLUSIONS: This study documents that stable rep-cap cells lines are severely hampered for rAAV assembly when a replicative adenovirus is substituted with an adenoviral plasmid. Furthermore, our results also suggest that the lack of amplification of the rep-cap genes, eventually combined with the altered distribution of the adenoviral proteins, E4orf6 and E1B-55kDa, is related to the low rAAV yields observed under these conditions.

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8(+) cells. IFN-γ-producing cells were also observed among effector memory CD4(+) cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.

The ultimate goal of liver-directed gene therapy for genetic diseases is the stable expression of a therapeutic transgene in a significant proportion of hepatocytes. This article considers the various liver-directed gene transfer procedures studied so far. Performances and limitations of currently available vector systems are discussed with respect to their clinical relevance. Although some improvements have been reported, naked DNA and nonviral gene transfer vectors induce transient expression in only a limited number of cells. Clinical applications of retrovirus-mediated gene transfer are hampered by the need to induce hepatocyte division. First-generation adenovirus vectors are highly efficient; however, they induce an immune response leading to the rapid rejection of transduced cells. Promising new vector systems have emerged, including gutless adenovirus vectors, adenoassociated vectors, and lentivirus vectors. However, these systems are still poorly documented and their relevance to liver-directed gene therapy must be confirmed. The present article summarizes current knowledge about gene transfer vectors designed for liver-directed gene therapy. We describe the advantages and drawbacks of viral and nonviral gene delivery vehicles that have been used to transfer genes to hepatocytes in experimental models. We analyze future trends that may lead to improved vector efficiency and ultimately to their proposed use in clinical trials.

Objective To provide new insights into the FOXG1- related clinical and imaging phenotypes and refine the phenotype-genotype correlation in FOXG1 syndrome. Methods We analyzed the clinical and imaging phenotypes of a cohort of 45 patients with a pathogenic or likely pathogenic FOXG1 variant and performed phenotype-genotype correlations. Results A total of 37 FOXG1 different heterozygous mutations were identified, of which 18 are novel. We described a broad spectrum of neurodevelopmental phenotypes, characterized by severe postnatal microcephaly and developmental delay accompanied by a hyperkinetic movement disorder, stereotypes and sleep disorders, and epileptic seizures. Our data highlighted 3 patterns of gyration, including frontal pachygyria in younger patients (26.7%), moderate simplified gyration (24.4%) and mildly simplified or normal gyration (48.9%), corpus callosum hypogenesis mostly in its frontal part, combined with moderate-to-severe myelination delay that improved and normalized with age. Frameshift and nonsense mutations in the N-terminus of FOXG1 , which are the most common mutation types, show the most severe clinical features and MRI anomalies. However, patients with recurrent frameshift mutations c.460dupG and c.256dupC had variable clinical and imaging presentations. Conclusions These findings have implications for genetic counseling, providing evidence that N-terminal mutations and large deletions lead to more severe FOXG1 syndrome, although genotype-phenotype correlations are not necessarily straightforward in recurrent mutations. Together, these analyses support the view that FOXG1 syndrome is a specific disorder characterized by frontal pachygyria and delayed myelination in its most severe form and hypogenetic corpus callosum in its milder form.

The purpose of our study was to evaluate lentiviral vector-mediated rat retinal transduction using simian immunodeficiency virus (SIV) pseudotyped with envelope proteins from vesicular stomatitis virus G glycoprotein (VSV-G), Mokola virus G protein (MK-G), amphotropic murine leukemia virus envelope (4070A-Env), influenza A virus hemagglutinin (HA), lymphocytic choriomeningitis virus G protein (LCMV-G), and RD114 retrovirus envelope (RD114-Env). The six pseudotyped lentivirus vectors carried CMV-driven green fluorescent protein (GFP) or beta-galactosidase (beta-gal) reporter genes. Intravitreal and subretinal injections of each pseudotyped recombinant SIV were performed in cohorts of Wistar rats. Our results showed that no transgene expression was detected after intravitreal injection of each pseudotyped SIV vector. Also, no transduction could be detected following subretinal injection of RD114 pseudotyped SIV vectors. However, selective transduction of retinal pigment epithelium (RPE) cells was repeatedly obtained after subretinal delivery of VSV-G, MK-G, 4070A-Env, HA, and LCMV-G pseudotyped SIV. GFP expression was maximum as soon as 4 days postadministration for VSV-G, MK-G, 4070A-Env, and HA pseudotypes, with no evidence of pseudotransduction for VSV-G. Maximum transgene expression was observed 3 weeks postinjection for LCMV-6. Importantly, HA and VSV-G pseudotyped SIV lead to such a high level of transgene expression that GFP-related toxicity occurred. Therefore, when a high level of GFP synthesis is achieved, replacement of enhanced GFP (egfp, Aequorea victoria) by a low-toxicity GFP (Renilla reniformis) cDNA is necessary to allow long-term expression.

Charcot-Marie-Tooth disease 1 A (CMT1A) results from a duplication of the PMP22 gene in Schwann cells and a deficit of myelination in peripheral nerves. Patients with CMT1A have reduced nerve conduction velocity, muscle wasting, hand and foot deformations and foot drop walking. Here, we evaluate the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9) expressing GFP and shRNAs targeting Pmp22 mRNA in animal models of Charcot-Marie-Tooth disease 1 A. Intra-nerve delivery of AAV2/9 in the sciatic nerve allowed widespread transgene expression in resident myelinating Schwann cells in mice, rats and non-human primates. A bilateral treatment restore expression levels of PMP22 comparable to wild-type conditions, resulting in increased myelination and prevention of motor and sensory impairments over a twelve-months period in a rat model of CMT1A. We observed limited off-target transduction and immune response using the intra-nerve delivery route. A combination of previously characterized human skin biomarkers is able to discriminate between treated and untreated animals, indicating their potential use as part of outcome measures.

Here we describe the development of a two-step chromatography process based on the use of ion-exchange resins for the purification of recombinant adeno-associated virus (rAAV) serotypes-2 and -5. In vitro and in vivo results demonstrate that this method, which does not require any prepurification step of the cell lysate, can be applied to obtain highly pure rAAV2 and rAAV5 stocks. As such, this procedure can be easily transferred in vector cores and also scaled up, allowing the direct comparison of these two, and potentially other, AAV serotypes in large animal models.

The question whether hepatocellular carcinoma (HCC) arises from dedifferentiation of mature hepatocytes or from proliferation of liver stem cells is still debated. In the present study, we used retroviral-mediated genetic labeling to investigate the fate of mature hepatocytes in rats after administration of diethylnitrosamine (DEN). Mature hepatocytes were genetically labeled by intravenous injection of retroviral vectors containing the Escherichia coli beta-galactosidase gene coupled to a nuclear localization signal (nls-LacZ) 1 day after partial hepatectomy. Liver biopsies performed after completion of hepatic regeneration showed that 18.3% of hepatocytes expressed the nls-LacZ transgene. Rats were then treated with DEN in drinking water for 12 weeks and sacrificed between 98 and 151 days after the onset of DEN administration. Clones of beta-galactosidase positive cells were observed, half of which (53%) also expressed the placental form of glutathione-S-transferase (GSTp), a marker of preneoplastic cells. HCCs of various sizes expressing GSTp were present in all animals. Careful examination of 90 HCCs revealed that 16 (17.7%) also expressed nls-LacZ. This figure precisely matched the proportion of labeled hepatocytes before DEN treatment (18.3%). In conclusion, a random clonal origin of HCC from mature hepatocytes is seen in the DEN model of hepatocarcinogenesis.

Here we describe the development of a two-step chromatography process based on the use of ion-exchange resins for the purification of recombinant adeno-associated virus (rAAV) serotypes-2 and-5. In vitro and in vivo results demonstrate that this method, which does not require any prepurification step of the cell lysate, can be applied to obtain highly pure rAAV2 and rAAV5 stocks. As such,this procedure can be easily transferred in vector cores and also scaled up, allowing the direct comparison of these two, and potentially other, AAV serotypes in large animal models.