Design of experiments as a decision tool for cell therapy manufacturing.

BACKGROUND AIMS: Cell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost. METHODS: Borrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. RESULTS: Using gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50 000-750 000 vg/cell AAV and 0.8-4 µg sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193 000 vg/cell AAV and 1.78 µg sgRNA. CONCLUSIONS: The authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics.

Safety Assessment of AAV2-hGDNF Administered Via Intracerebral Injection in Rats for Treatment of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective biologic in Parkinson's disease models. Adeno-associated viral vector serotype 2 (AAV2)-human GDNF safety was assessed in rats treated with a single intracerebral dose of vehicle, 6.8 × 108, 6.8 × 109, or 5.2 × 1010 vector genomes (vg)/dose followed by interim sacrifices on day 7, 31, 90, and 376. There were no treatment-related effects observed on food consumption, body weight, hematology, clinical chemistry, coagulation parameters, neurobehavioral parameters, organ weights, or serum GDNF and anti-GDNF antibody levels. Increased serum anti-AAV2 neutralizing antibody titers were observed in the 5.2 × 1010 vg/dose group. Histopathological lesions were observed at the injection site in the 6.8 × 109 vg/dose (day 7) and 5.2 × 1010 vg/dose groups (days 7 and 31) and consisted of gliosis, mononuclear perivascular cuffing, intranuclear inclusion bodies, and/or apoptosis on day 7 and mononuclear perivascular cuffing on day 31. GDNF immunostaining was observed in the injection site in all dose groups through day 376 indicating no detectable impacts of anti-AAV2 neutralizing antibody. There was no evidence of increased expression of calcitonin gene-related peptide or Swann cell hyperplasia in the cervical and lumbar spinal cord or medulla oblongata at the 5.2 × 1010 vg/dose level indicating lack of hyperplastic effects. In conclusion, no systemic toxicity was observed, and the local toxicity observed at the injection site appeared to be reversible demonstrating a promising safety profile of intracerebral AAV2-GDNF delivery. Furthermore, an intracerebral dose of 6.8 × 108 AAV2-GDNF vg/dose was considered to be a no observed adverse effect level in rats.

Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant.

BACKGROUND: The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. METHODS: We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX-R338L) transgene at a dose of 5×1011 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. RESULTS: No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady-state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P=0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P=0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. CONCLUSIONS: We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092 .).

Efficacy, Safety, and Durability of Voretigene Neparvovec-rzyl in RPE65 Mutation-Associated Inherited Retinal Dystrophy: Results of Phase 1 and 3 Trials.

PURPOSE: To report the durability of voretigene neparvovec-rzyl (VN) adeno-associated viral vector-based gene therapy for RPE65 mutation-associated inherited retinal dystrophy (IRD), including results of a phase 1 follow-on study at year 4 and phase 3 study at year 2. DESIGN: Open-label phase 1 follow-on clinical trial and open-label, randomized, controlled phase 3 clinical trial. PARTICIPANTS: Forty subjects who received 1.5×1011 vector genomes (vg) of VN per eye in at least 1 eye during the trials, including 11 phase 1 follow-on subjects and 29 phase 3 subjects (20 original intervention [OI] and 9 control/intervention [CI]). METHODS: Subretinal injection of VN in the second eye of phase 1 follow-on subjects and in both eyes of phase 3 subjects. MAIN OUTCOME MEASURES: End points common to the phase 1 and phase 3 studies included change in performance on the Multi-Luminance Mobility Test (MLMT) within the illuminance range evaluated, full-field light sensitivity threshold (FST) testing, and best-corrected visual acuity (BCVA). Safety end points included adverse event reporting, ophthalmic examination, physical examination, and laboratory testing. RESULTS: Mean (standard deviation) MLMT lux score change was 2.4 (1.3) at 4 years compared with 2.6 (1.6) at 1 year after administration in phase 1 follow-on subjects (n = 8), 1.9 (1.1) at 2 years, and 1.9 (1.0) at 1 year post-administration in OI subjects (n = 20), and 2.1 (1.6) at 1 year post-administration in CI subjects (n = 9). All 3 groups maintained an average improvement in FST, reflecting more than a 2 log10(cd.s/m2) improvement in light sensitivity at 1 year and subsequent available follow-up visits. The safety profile was consistent with vitrectomy and the subretinal injection procedure, and no deleterious immune responses occurred. CONCLUSIONS: After VN gene augmentation therapy, there was a favorable benefit-to-risk profile with similar improvement demonstrated in navigational ability and light sensitivity among 3 groups of subjects with RPE65 mutation-associated IRD, a degenerative disease that progresses to complete blindness. The safety profile is consistent with the administration procedure. These data suggest that this effect, which is nearly maximal by 30 days after VN administration, is durable for 4 years, with observation ongoing.

Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery.

This study evaluated the safety and tolerability of ocular RS1 adeno-associated virus (AAV8-RS1) gene augmentation therapy to the retina of participants with X-linked retinoschisis (XLRS). XLRS is a monogenic trait affecting only males, caused by mutations in the RS1 gene. Retinoschisin protein is secreted principally in the outer retina, and its absence results in retinal cavities, synaptic dysfunction, reduced visual acuity, and susceptibility to retinal detachment. This phase I/IIa single-center, prospective, open-label, three-dose-escalation clinical trial administered vector to nine participants with pathogenic RS1 mutations. The eye of each participant with worse acuity (≤63 letters; Snellen 20/63) received the AAV8-RS1 gene vector by intravitreal injection. Three participants were assigned to each of three dosage groups: 1e9 vector genomes (vg)/eye, 1e10 vg/eye, and 1e11 vg/eye. The investigational product was generally well tolerated in all but one individual. Ocular events included dose-related inflammation that resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in a dose-related fashion, but no antibodies against RS1 were observed. Retinal cavities closed transiently in one participant. Additional doses and immunosuppressive regimens are being explored to pursue evidence of safety and efficacy (ClinicalTrials.gov: NCT02317887).

Progress and challenges in viral vector manufacturing.

Promising results in several clinical studies have emphasized the potential of gene therapy to address important medical needs and initiated a surge of investments in drug development and commercialization. This enthusiasm is driven by positive data in clinical trials including gene replacement for Hemophilia B, X-linked Severe Combined Immunodeficiency, Leber's Congenital Amaurosis Type 2 and in cancer immunotherapy trials for hematological malignancies using chimeric antigen receptor T cells. These results build on the recent licensure of the European gene therapy product Glybera for the treatment of lipoprotein lipase deficiency. The progress from clinical development towards product licensure of several programs presents challenges to gene therapy product manufacturing. These include challenges in viral vector-manufacturing capacity, where an estimated 1-2 orders of magnitude increase will likely be needed to support eventual commercial supply requirements for many of the promising disease indications. In addition, the expanding potential commercial product pipeline and the continuously advancing development of recombinant viral vectors for gene therapy require that products are well characterized and consistently manufactured to rigorous tolerances of purity, potency and safety. Finally, there is an increase in regulatory scrutiny that affects manufacturers of investigational drugs for early-phase clinical trials engaged in industry partnerships. Along with the recent increase in biopharmaceutical funding in gene therapy, industry partners are requiring their academic counterparts to meet higher levels of GMP compliance at earlier stages of clinical development. This chapter provides a brief overview of current progress in the field and discusses challenges in vector manufacturing.

Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial.

BACKGROUND: Phase 1 studies have shown potential benefit of gene replacement in RPE65-mediated inherited retinal dystrophy. This phase 3 study assessed the efficacy and safety of voretigene neparvovec in participants whose inherited retinal dystrophy would otherwise progress to complete blindness. METHODS: In this open-label, randomised, controlled phase 3 trial done at two sites in the USA, individuals aged 3 years or older with, in each eye, best corrected visual acuity of 20/60 or worse, or visual field less than 20 degrees in any meridian, or both, with confirmed genetic diagnosis of biallelic RPE65 mutations, sufficient viable retina, and ability to perform standardised multi-luminance mobility testing (MLMT) within the luminance range evaluated, were eligible. Participants were randomly assigned (2:1) to intervention or control using a permuted block design, stratified by age (<10 years and ≥10 years) and baseline mobility testing passing level (pass at ≥125 lux vs <125 lux). Graders assessing primary outcome were masked to treatment group. Intervention was bilateral, subretinal injection of 1·5 × 1011 vector genomes of voretigene neparvovec in 0·3 mL total volume. The primary efficacy endpoint was 1-year change in MLMT performance, measuring functional vision at specified light levels. The intention-to-treat (ITT) and modified ITT populations were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, number NCT00999609, and enrolment is complete. FINDINGS: Between Nov 15, 2012, and Nov 21, 2013, 31 individuals were enrolled and randomly assigned to intervention (n=21) or control (n=10). One participant from each group withdrew after consent, before intervention, leaving an mITT population of 20 intervention and nine control participants. At 1 year, mean bilateral MLMT change score was 1·8 (SD 1·1) light levels in the intervention group versus 0·2 (1·0) in the control group (difference of 1·6, 95% CI 0·72-2·41, p=0·0013). 13 (65%) of 20 intervention participants, but no control participants, passed MLMT at the lowest luminance level tested (1 lux), demonstrating maximum possible improvement. No product-related serious adverse events or deleterious immune responses occurred. Two intervention participants, one with a pre-existing complex seizure disorder and another who experienced oral surgery complications, had serious adverse events unrelated to study participation. Most ocular events were mild in severity. INTERPRETATION: Voretigene neparvovec gene replacement improved functional vision in RPE65-mediated inherited retinal dystrophy previously medically untreatable. FUNDING: Spark Therapeutics.

Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial.

BACKGROUND: Safety and efficacy have been shown in a phase 1 dose-escalation study involving a unilateral subretinal injection of a recombinant adeno-associated virus (AAV) vector containing the RPE65 gene (AAV2-hRPE65v2) in individuals with inherited retinal dystrophy caused by RPE65 mutations. This finding, along with the bilateral nature of the disease and intended use in treatment, prompted us to determine the safety of administration of AAV2-hRPE65v2 to the contralateral eye in patients enrolled in the phase 1 study. METHODS: In this follow-on phase 1 trial, one dose of AAV2-hRPE65v2 (1.5 × 10(11) vector genomes) in a total volume of 300 µL was subretinally injected into the contralateral, previously uninjected, eyes of 11 children and adults (aged 11-46 years at second administration) with inherited retinal dystrophy caused by RPE65 mutations, 1.71-4.58 years after the initial subretinal injection. We assessed safety, immune response, retinal and visual function, functional vision, and activation of the visual cortex from baseline until 3 year follow-up, with observations ongoing. This study is registered with ClinicalTrials.gov, number NCT01208389. FINDINGS: No adverse events related to the AAV were reported, and those related to the procedure were mostly mild (dellen formation in three patients and cataracts in two). One patient developed bacterial endophthalmitis and was excluded from analyses. We noted improvements in efficacy outcomes in most patients without significant immunogenicity. Compared with baseline, pooled analysis of ten participants showed improvements in mean mobility and full-field light sensitivity in the injected eye by day 30 that persisted to year 3 (mobility p=0.0003, white light full-field sensitivity p<0.0001), but no significant change was seen in the previously injected eyes over the same time period (mobility p=0.7398, white light full-field sensitivity p=0.6709). Changes in visual acuity from baseline to year 3 were not significant in pooled analysis in the second eyes or the previously injected eyes (p>0.49 for all time-points compared with baseline). INTERPRETATION: To our knowledge, AAV2-hRPE65v2 is the first successful gene therapy administered to the contralateral eye. The results highlight the use of several outcome measures and help to delineate the variables that contribute to maximal benefit from gene augmentation therapy in this disease. FUNDING: Center for Cellular and Molecular Therapeutics at The Children's Hospital of Philadelphia, Spark Therapeutics, US National Institutes of Health, Foundation Fighting Blindness, Institute for Translational Medicine and Therapeutics, Research to Prevent Blindness, Center for Advanced Retinal and Ocular Therapeutics, Mackall Foundation Trust, F M Kirby Foundation, and The Research Foundation-Flanders.

Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

AAV-mediated expression of an ADAMTS13 variant prevents shigatoxin-induced thrombotic thrombocytopenic purpura.

Severe deficiency of plasma ADAMTS13 activity causes thrombotic thrombocytopenic purpura (TTP), a life-threatening syndrome for which plasma is the only effective therapy currently available. As much as 5% of TTP cases are hereditary, resulting from mutations of the ADAMTS13 gene. Here, we report the efficacy and safety of recombinant adeno-associated virus serotype 8 (AAV8)-mediated expression of a murine ADAMTS13 variant (MDTCS), truncated after the spacer domain, in a murine model of TTP. Administration of AAV8-hAAT-mdtcs at doses greater than 2.6 × 10(11) vg/kg body weight resulted in sustained expression of plasma ADAMTS13 activity at therapeutic levels. Expression of the truncated ADAMTS13 variant eliminated circulating ultralarge von Willebrand factor multimers, prevented severe thrombocytopenia, and reduced mortality in Adamts13(-/-) disease-prone mice triggered by shigatoxin-2. These data support AAV vector-mediated expression of a comparable truncated ADAMTS13 variant as a novel therapeutic approach for hereditary TTP in humans.

Cerebral infusion of AAV9 vector-encoding non-self proteins can elicit cell-mediated immune responses.

There is considerable interest in the use of adeno-associated virus serotype 9 (AAV9) for neurological gene therapy partly because of its ability to cross the blood-brain barrier to transduce astrocytes and neurons. This raises the possibility that AAV9 might also transduce antigen-presenting cells (APC) in the brain and provoke an adaptive immune response. We tested this hypothesis by infusing AAV9 vectors encoding foreign antigens, namely human aromatic L-amino acid decarboxylase (hAADC) and green fluorescent protein (GFP), into rat brain parenchyma. Over ensuing weeks, both vectors elicited a prominent inflammation in transduced brain regions associated with upregulation of MHC II in glia and associated lymphocytic infiltration. Transduction of either thalamus or striatum with AAV9-hAADC evinced a significant loss of neurons and induction of anti-hAADC antibodies. We conclude that AAV9 transduces APC in the brain and, depending on the immunogenicity of the transgene, can provoke a full immune response that mediates significant brain pathology. We emphasize, however, that these observations do not preclude the use of AAV serotypes that can transduce APC. However, it does potentially complicate preclinical toxicology studies in which non-self proteins are expressed at a level sufficient to trigger cell-mediated and humoral immune responses.

Three-year follow-up after unilateral subretinal delivery of adeno-associated virus in patients with Leber congenital Amaurosis type 2.

OBJECTIVE: The aim of this study was to show the clinical data of long-term (3-year) follow-up of 5 patients affected by Leber congenital amaurosis type 2 (LCA2) treated with a single unilateral injection of adeno-associated virus AAV2-hRPE65v2. DESIGN: Clinical trial. PARTICIPANTS: Five LCA2 patients with RPE65 gene mutations. METHODS: After informed consent and confirmation of trial eligibility criteria, the eye with worse visual function was selected for subretinal delivery of adeno-associated virus (AAV2-hRPE65v2). Subjects were evaluated before and after surgery at designated follow-up visits (1, 2, 3, 14, 30, 60, 90, 180, 270, and 365 days, 1.5 years, and 3 years) by complete ophthalmic examination. Efficacy for each subject was monitored with best-corrected visual acuity, kinetic visual field, nystagmus testing, and pupillary light reflex. MAIN OUTCOME MEASURES: Best-corrected visual acuity, kinetic visual field, nystagmus testing, and pupillary light reflex. RESULTS: The data showed a statistically significant improvement of best-corrected visual acuity between baseline and 3 years after treatment in the treated eye (P<0.001). In all patients, an enlargement of the area of visual field was observed that remained stable until 3 years after injection (average values: baseline, 1058 deg(2) vs. 3 years after treatment, 4630 deg(2)) and a reduction of the nystagmus frequency compared with baseline at the 3-year time point. Furthermore, a statistically significant difference was observed in the pupillary constriction of the treated eye (P<0.05) compared with the untreated eye in 3 patients at 1- and 3-year time points. No patients experienced serious adverse events related to the vector in the 3-year postinjection period. CONCLUSIONS: The long-term follow-up data (3 years) on the 5-patient Italian cohort involved in the LCA2 gene therapy clinical trial clearly showed a stability of improvement in visual and retinal function that had been achieved a few months after treatment. Longitudinal data analysis showed that the maximum improvement was achieved within 6 months after treatment, and the visual improvement was stable up to the last observed time point. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Assessing the potential for AAV vector genotoxicity in a murine model.

Gene transfer using adeno-associated virus (AAV) vectors has great potential for treating human disease. Recently, questions have arisen about the safety of AAV vectors, specifically, whether integration of vector DNA in transduced cell genomes promotes tumor formation. This study addresses these questions with high-dose liver-directed AAV-mediated gene transfer in the adult mouse as a model (80 AAV-injected mice and 52 controls). After 18 months of follow-up, AAV-injected mice did not show a significantly higher rate of hepatocellular carcinoma compared with controls. Tumors in mice treated with AAV vectors did not have significantly different amounts of vector DNA compared with adjacent normal tissue. A novel high-throughput method for identifying AAV vector integration sites was developed and used to clone 1029 integrants. Integration patterns in tumor tissue and adjacent normal tissue were similar to each other, showing preferences for active genes, cytosine-phosphate-guanosine islands, and guanosine/cytosine-rich regions. [corrected] Gene expression data showed that genes near integration sites did not show significant changes in expression patterns compared with genes more distal to integration sites. No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation.

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.

We have previously shown that 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 B. We carried out a phase 1/2 dose-escalation clinical study 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 10(12) 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 approximately 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. We conclude that 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.

Pharmacological modulation of humoral immunity in a nonhuman primate model of AAV gene transfer for hemophilia B.

Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a "single-shot " therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.