Lentiviral Vector-based Gene Therapy Research Articles

Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the β-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott–Aldrich syndrome

Patients with Wiskott–Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Long-Term Follow-up Study after Lentiviral Hematopoietic Stem/Progenitor Cell Gene Therapy for Wiskott - Aldrich Syndrome

▪Wiskott Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency associated with thrombocytopenia, eczema, infectious, autoimmune complications, and lymphomas. Patients lacking an HLA-matched donor may benefit from an alternative therapeutic approach based on the infusion of autologous gene corrected CD34+ cells.We previously reported a non-randomised, open-label, phase 1/2 clinical study applying a lentiviral vector based gene therapy (GT) protocol in 7 paediatric patients with severe WAS (score ≥ 3/5) (S. Hacein-Bey Abina et al, JAMA 2015). One patient died 7 months after GT because of pre-existing severe opportunistic infections, as reported. Two additional patients have been treated since that initial report, with a follow-up of at least 4 years. We here present a comprehensive long-term study on 8 patients with a follow-up from 4 to 9 years (median 7.6). The safety and efficacy of the approach is thoroughly investigated, with a particular focus on the correction of thrombocytopenia and auto-immunity.A stable engraftment of genetically and functionally corrected lymphoid and myeloid cells was reached in all patients, with no serious treatment-associated adverse events or concerning clonal expansion. Corrected lymphoid cells displayed a selective advantage over time with increasing vector copy number (VCN) level. In turn, this led to (i) sustained expression of WAS protein (WASp) in the patients' cells and (ii) clinical resolution of severe eczema and susceptibility to recurrent infections. In line with these results, T-cell function was restored after GT, as shown by the recovery of immune synapse assembly and the normalization of naïve T cell numbers. The T-cell compartment was also reconstituted in the patient treated at the age of 30 years, suggesting that GT for WAS is a treatment option in adult patients.In parallel with the robustness of T-cell reconstitution a normalized B-cell compartment was observed after GT, as shown in particular by increasing levels of WASp + switched memory B cells over time and the age-matched levels of KRECs. Five patients out of 8 were able to discontinue Ig replacement therapy while achieving normal post-vaccination antibody titers.Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. After GT, a few autoimmune manifestations were observed: the persistence of lower extremity vasculitis (P2, very severe prior to GT), the new occurrence of nephrotic syndrome (P9), and the presence of anti-platelet antibodies (P2, P4, P7). The levels of circulating autoantibodies detected before GT (including ANA and vasculitis-related autoantibodies) normalized after treatment. Following GT, platelets were found to express sub-normal levels of WASp and to only partially augment their size. Platelet function studies indicated a partial correction of the platelet compartment achieved by GT, which may be sufficient to prevent occurrence of the hemorrhagic symptoms typical of WAS.Our results suggest that lentiviral GT provides sustained clinical benefits for patients with WAS. Overall clinical remission was observed in our patients despite very severe disease scores before GT. More efficacious and more reliable transduction protocols and conditioning regimen are likely to further improve outcomes, particularly with regard to platelet recovery, where the advantages of intrinsic correction are less apparent. DisclosuresBooth: Orchard Therapeutics: Consultancy, Honoraria; SOBI: Consultancy, Honoraria; Takeda: Honoraria; GSK: Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cavazzana: Smart Immune: Other: co-founder.

Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown, β-Globin Induction and Simultaneous Repression of β-Globin for the Potential Treatment of Sickle Cell Disease

▪Ex vivo lentiviral vector-based gene therapy has shown promise as a treatment for severe sickle cell disease (SCD) (Ribeil, NEJM 2017). This approach relies on the regulated expression of a therapeutic anti-sickling β-globin (T87Q) at sufficient levels to reduce or prevent the polymerization of mutant hemoglobin S (HbS). Reactivation of fetal hemoglobin (HbF), by reducing expression of BCL11A with a lineage-specific miRNA adapted shRNA (shRNA (miR)), is an alternative strategy to generate therapeutic hemoglobin in transduced cells (Brendel, JCI 2016). Erythroid-specific silencing of BCL11A has an added benefit of simultaneously reducing expression of adult β-globin (which is mutated to HbS in SCD). The resulting increase in HbF (which itself has anti-sickling properties) and concomitant reduction in HbS may provide an extremely powerful approach to attenuating the sickling tendency of red blood cells. Here we provide new preclinical data generated to support the initiation of a first-in-human trial studying BCL11A silencing. Specifically, we report the characterization of a lentiviral vector construct encoding an erythroid-specific BCL11A shRNA(miR), termed BCH_BBLCR-shRNA(miR), adapted from our previously reported vector, that was developed to overcome poor vector titers observed in the manufacturing scale-up of our original vector. Healthy donor human hematopoietic stem cells transduced with lentiviral vector BCH_BBLCR-shRNA(miR) produced at scale under GMP conditions achieved high vector copy numbers (VCNs; >5 copies/diploid genome) and a 5-fold higher induction of HbF than seen with mock transduced cells (65.5% vs 13.1%). SCD human HSCs transduced with high-titer BCH_BBLCR-shRNA(miR) vector achieved >80% transduction efficiency, resulting in HbF induction between 70% and 81% of all β-like globin chains (with concomitant suppression of HbS, with residual HbS ranging from 19% to 30%). In addition, we observed no impact on in vitro growth and/or differentiation of erythroid cells derived from CD34+ cells. Analysis of individual colonies showed that >96% of individual BFU-E cells have HbF >50%. Repopulation studies in NSG mice transplanted with healthy donor human HSCs transduced (>80%) with BCH_BBLCR-shRNA(miR) showed no deleterious effects in vivo of targeting BCL11A in a lineage-specific fashion even at higher VCN (>3 copies/diploid genome). Lastly, consistent with the expected behavior of current generation lentiviral vectors, in vitro immortalization assays confirmed that the BCH_BBLCR-shRNA(miR) vector did not induce immortalization in this setting. Together these data demonstrate that LVV BCH_BBLCR-shRNA(miR) is (i) safe and efficacious in preclinical studies, and (ii) can be generated at scale in a GMP setting to support a clinical study of this potentially attractive approach to the treatment of SCD. DisclosuresNegre:bluebird bio: Employment, Equity Ownership. Parsons:bluebird bio: Employment, Equity Ownership. Luo:bluebird bio Inc.: Employment. Christiansen:bluebird bio: Employment, Equity Ownership. Lewis:bluebird bio: Employment, Equity Ownership. Veres:bluebird bio: Employment, Equity Ownership. Bonner:bluebird bio: Employment, Equity Ownership. Williams:bluebird bio: Patents & Royalties: Licensed IP to bluebird, Research Funding.

Long-Term Follow-Up of a Phase I/II Study of ProSavin, a Lentiviral Vector Gene Therapy for Parkinson's Disease

Parkinson's disease is typically treated with oral dopamine replacement therapies. However, long-term use is complicated by motor fluctuations from intermittent stimulation of dopamine receptors and off-target effects. ProSavin, a lentiviral vector based gene therapy that delivers local and continuous dopamine, was previously shown to be well tolerated in a Phase I/II first-in-human study, with significant improvements in motor behavior from baseline at 1 year. Here, patients with Parkinson's disease from the open-label trial were followed up in the long term to assess the safety and efficacy of ProSavin after bilateral injection into the putamen. Fifteen patients who were previously treated with ProSavin have been followed for up to 5 years, with some having been seen for 8 years. Eight patients received deep brain stimulation at different time points, and their subsequent assessments continued to assess safety. Ninety-six drug-related adverse events were reported (87 mild, 6 moderate, 3 severe) of which more than half occurred in the first year. The most common drug-related events were dyskinesias (33 events, 11 patients) and on–off phenomena (22 events, 11 patients). A significant improvement in the defined “off” Unified Parkinson's Disease Rating Scale part III motor scores, compared to baseline, was seen at 2 years (mean score 29 · 2 vs. 38 · 4, n = 14, p < 0.05) and at 4 years in 8/15 patients. ProSavin continued to be safe and well tolerated in patients with Parkinson's disease. Moderate improvements in motor behavior over baseline continued to be reported in the majority of patients who could still be evaluated up to 5 years of follow-up.

Gene Therapy: Where We Started and Where the Field Is Going

In the more than 20 years since the first successful gene transfer was performed in a human subject (Blaese, R.M. et al. Science 1995; 270, 475-480), the promise of gene therapy has been at once tantalizingly close and just out of reach. Today, gene-modified therapies are being used to treat a variety of diseases, and direct manipulation of the human genome for therapeutic effect is becoming a reality. While many advances in gene-based therapeutics have come in the oncology space, non-malignant hematologic and primary immune disorders remain important targets for these approaches. The ability to manipulate hematopoietic stem cells (HSCs) ex vivo and return the modified HSCs to the patient offers many potential advantages over allogeneic HSC transplantation. Additionally, the potential to introduce new genes with therapeutic potential, or to modify genes to modulate protein expression, may open new avenues for transformative therapies for genetic diseases. The progress of gene therapy for hemoglobinopathies - from the γ-retroviral vector technology that established the field, to the accumulating clinical experience with lentiviral vector-based gene therapy and the potential for gene editing-based approaches to address these diseases - provides insight into the development of genetic therapeutics. Transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) result from pathogenic mutations in the β-globin gene, HBB . Addition of functional HBB genes into autologous hematopoietic stem cells has the potential to offer the long-term therapeutic benefits of allogeneic HSC transplantation without the complications of graft vs host disease. The first human proof of concept of this approach came in a study of the HPV569 lentiviral vector coding for therapeutic β-globin, which was successfully introduced into the HSCs of patients with TDT and resulted in sustained clinical benefit in some patients (Cavazzana-Calvo et al., Nature 2010; 467(7313):318-22). Beyond restoring normal β-globin production, studies with HPV569 and its improved variant BB305, have shown that it is possible to drive expression of a β-globin variant with a point mutation, T87Q, designed to mimic the anti-sickling effect of γ-globin. This innovation may have important implications for patients with SCD, where introduction of normal β-globin may be insufficient to ameliorate the red blood cell sickling and polymerization that can cause painful and damaging vaso-occlusive crises. It has taken more than 20 years for HSC gene addition to reach safety and efficacy thresholds that may allow it to be used routinely for patients with hemoglobinopathies, but this approach is now nearing maturity. Important refinements in LVV architecture and advances in HBB gene cassette design have yielded promising results in multiple clinical studies of TDT and SCD (e.g. Ribeil J.A. et al., N Engl J Med 2017; 376:848-855). There is also significant excitement about the potential for gene editing approaches to address the hemoglobinopathies even as these technologies are just beginning to transition from lab to clinic. Critical questions of both efficacy and safety remain regarding the path forward for nuclease-based editing technologies such as CRISPR, ZFN, and megaTALs. Key lessons from the development of clinical gene addition therapies in the hemoglobinopathies may help chart the path forward for gene editing technologies. Disclosures Gregory: bluebirdbio: Employment; Merck KGaA: Membership on an entity's Board of Directors or advisory committees.

A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies.

Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations and how they are administered in the United Kingdom, although many of the principles will be similar for other regions, including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarizes the extant regulatory guidance for gene therapies, categorized as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy.

A guide to approaching regulatory considerations for lentiviral-mediated gene therapies

Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well-characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is non-pathogenic and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations, and how they are administered in the United Kingdom, although many of the principles will be similar for other regions including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarises the extant regulatory guidance for gene therapies, categorised as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy.

Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects.

Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)‐induced central nervous system damage. However, all HSPCT‐treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor‐derived cells and 2) insufficient uptake of donor cell‐secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation‐independent mannose 6‐phosphate‐receptor (CI‐MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu‐mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell‐specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT‐mediated correction of GALC deficiency in target cells expressing low levels of CI‐MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose‐6‐phosphate‐independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects: BMT-Lentiviral Vectors Therapy for Krabbe's Disease

Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects: BMT-Lentiviral Vectors Therapy for Krabbe's Disease

A New Gene Therapy for Parkinson Disease

Parkinson disease (PD) is a progressive neurodegenerative syndrome that affects motor and nonmotor abilities. This manufacturer-funded study addressed the safety, tolerability, and efficacy of bilateral intrastriatal delivery of ProSavin, a lentiviral vector–based gene therapy intended to enhance continuous dopamine production in the brain. The study was a phase 1/2 open-label trial with an open-label follow-up for safety. Fifteen patients with mild-to-moderate PD and a well-documented response to dopamine therapy received low-dose (1·9×107 transducing units [TU]; 3 patients), mid-dose …

Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial

Parkinson's disease is typically treated with oral dopamine replacement therapies; however, long-term treatment leads to motor complications and, occasionally, impulse control disorders caused by intermittent stimulation of dopamine receptors and off-target effects, respectively. We aimed to assess the safety, tolerability, and efficacy of bilateral, intrastriatal delivery of ProSavin, a lentiviral vector-based gene therapy aimed at restoring local and continuous dopamine production in patients with advanced Parkinson's disease. We undertook a phase 1/2 open-label trial with 12-month follow-up at two study sites (France and UK) to assess the safety and efficacy of ProSavin after bilateral injection into the putamen of patients with Parkinson's disease. All patients were then enrolled in a separate open-label follow-up study of long-term safety. Three doses were assessed in separate cohorts: low dose (1·9×10(7) transducing units [TU]); mid dose (4·0×10(7) TU); and high dose (1×10(8) TU). Inclusion criteria were age 48-65 years, disease duration 5 years or longer, motor fluctuations, and 50% or higher motor response to oral dopaminergic therapy. The primary endpoints of the phase 1/2 study were the number and severity of adverse events associated with ProSavin and motor responses as assessed with Unified Parkinson's Disease Rating Scale (UPDRS) part III (off medication) scores, at 6 months after vector administration. Both trials are registered at ClinicalTrials.gov, NCT00627588 and NCT01856439. 15 patients received ProSavin and were followed up (three at low dose, six mid dose, six high dose). During the first 12 months of follow-up, 54 drug-related adverse events were reported (51 mild, three moderate). Most common were increased on-medication dyskinesias (20 events, 11 patients) and on-off phenomena (12 events, nine patients). No serious adverse events related to the study drug or surgical procedure were reported. A significant improvement in mean UPDRS part III motor scores off medication was recorded in all patients at 6 months (mean score 38 [SD 9] vs 26 [8], n=15, p=0·0001) and 12 months (38 vs 27 [8]; n=15, p=0·0001) compared with baseline. ProSavin was safe and well tolerated in patients with advanced Parkinson's disease. Improvement in motor behaviour was observed in all patients. Oxford BioMedica.

306. Lentiviral Vector-Based Gene Therapy for Wiskott-Aldrich Syndrome: Preliminary Results from the French Center

306. Lentiviral Vector-Based Gene Therapy for Wiskott-Aldrich Syndrome: Preliminary Results from the French Center

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

Analysis of Lentiviral Vector Integration in HIV+ Study Subjects Receiving Autologous Infusions of Gene Modified CD4+ T Cells

Lentiviral vector-based gene therapy has been used to target the human immunodeficiency virus (HIV) using an antisense env payload. We have analyzed lentiviral-vector integration sites from three treated individuals. We compared integration sites from the ex vivo vector-transduced CD4+ cell products to sites from cells recovered at several times after infusion. Integration sites were analyzed using 454 pyrosequencing, yielding a total of 7,782 unique integration sites from the ex vivo product and 237 unique sites from cells recovered after infusion. Integrated vector copies in both data sets were found to be strongly enriched within active genes and near epigenetic marks associated with active transcription units. Analysis of integration relative to nucleosome structure on target DNA indicated favoring of integration in outward facing DNA major grooves on the nucleosome surface. There was no indication that growth of transduced cells after infusion resulted in enrichment for integration sites near proto-oncogene 5'-ends or within tumor suppressor genes. Thus, this first look at the longitudinal evolution of cells transduced with a lentiviral vector after infusion of gene modified CD4+ cells provided no evidence for abnormal expansions of cells due to vector-mediated insertional activation of proto-oncogenes.

375. A Glaucoma Gene Therapy Model: Eighteen Cat Study Revealing Long-Term Targeted Genetic Modification of the Anterior Chamber by Lentiviral Vectors

PURPOSE: Glaucoma is a promising candidate for lentiviral vector-based gene therapy, since its main cause, intraocular pressure elevation, results from pathophysiology that is i) chronic; ii) anatomically restricted to a discrete region in the anterior chamber, the trabecular meshwork (TM); iii) readily accessible via a transcorneal approach; and iv) frequently refractory to conventional therapies, such that glaucoma remains a leading cause of irreversible blindness. We present long-term results of transcorneal lentiviral vector injections in 18 domestic cats, which were studied as the most accessible large animal having anatomic similarity of the anterior chamber to the human eye. Animals were followed for 1.2 to 2.2 years. Both marker genes (eGFP) and wild-type and mutant versions of the myocilin gene, which has been implicated in uncommon forms of familial glaucoma, were studied. METHODS: 18 domestic cats received single bolus anterior chamber injections of FIV vector in doses of 106 (16 eyes) or 107 (19 eyes) transducing units (TU). Vectors encoded either eGFP, or a combination of feline myocilin (fMYOC or fMYOC with a Y423H mutation, equivalent to human MYOC Y437H) plus eGFP. A control vector lacked a gene in position 1. Follow-up included IOP measurements, slit lamp biomicroscopy, and gonioscopy. Eyes were then enucleated, examined with fluorescence microscopy, immunostaining for fMYOC and eGFP, and quantitative real-time vector PCR for vector DNA. RESULTS: Single gene eGFP vectors resulted in long-term, high-grade eGFP expression that was remarkably well targeted to the trabecular meshwork and was readily detected non-invasively for over two years. IRES-translated eGFP resulted in less eGFP expression in vitro and in vivo, and was detected in 29 of 32 eyes by gonioscope but was less than seen with translation initiated at the 5' cap site. However, considerably more extensive GFP expression was discovered when eyes were examined as dissected flat mounts after sacrifice. Expression of myocilins and eGFP was further confirmed by immunohistochemistry. Vector DNA copy number correlated with transgene expression. Mild AC inflammation was observed in some animals but resolved after 1|[ndash]|2 weeks. In all animals, IOP at end of study did not differ significantly from initial IOP (19|[ndash]|25 mmHg). CONCLUSIONS: Sustained, high-grade eGFP expression lasting over two years was achieved in TM. Vector-mediated eGFP fluorescence can be detected non-invasively via gonioscope, allowing real-time serial monitoring of transgene expression over time. This nevertheless underestimates long-term in vivo transgene expression, since considerably more eGFP expression was discovered after enucleation. Transduction of fMYOC or Y423H fMYOC did not cause elevated IOP. In summary, high level, long-term, and tissue- targeted gene expression suitable for glaucoma gene therapy is feasible with transcorneally-injected lentiviral vectors. Similar vector tests in macaques have been initiated.

Transduction of human glial and neuronal tumor cells with different lentivirus vector pseudotypes

Lentiviral vectors have proven to be valuable tools for in vitro and in vivo gene delivery because they can transduce dividing and non-dividing cells efficiently, and mediate long-term gene expression. Pseudotyping of lentiviral vectors with envelope proteins other than VSV-G has resulted in enhanced transduction of certain cell types and tissues. In order to improve lentiviral vector-based gene therapy for peripheral neuroectodermal and brain tumors, we compared the efficiency of eight different lentivirus pseudotypes in transducing neuronal and glial tumor cell lines. Here, lentiviral vectors pseudotyped with the envelopes from human foamy virus, rabies, Mokola or amphotropic murine leukemia virus displayed the highest transduction efficiency in neuroblastomas, whereas pseudotyping with the lymphocytic choriomeningitis virus glycoprotein from strain Armstrong 53b resulted in the highest transduction efficiency in gliomas.

Onco-retroviral and lentiviral vector-based gene therapy for hemophilia: preclinical studies.

Hemophilia A and B gene therapy requires long-term and stable expression of coagulation factor VIII (FVIII) or factor IX (FIX), respectively, and would need to compare favorably with protein replacement therapy. Onco-retroviral and lentiviral vectors are attractive vectors for gene therapy of hemophilia. These vectors have the potential for long-term expression because they integrate stably in the target cell genome. Whereas onco-retroviral vectors can only transduce dividing cells, lentiviral vectors can transduce a broad variety of cell types irrespective of cell division. Several preclinical and clinical studies have explored the use of onco-retroviral and, more recently, lentiviral vectors for gene therapy of hemophilia A or B. Both ex vivo and in vivo gene therapy approaches have been evaluated, resulting in therapeutic FVIII or FIX levels in preclinical animal models. Whereas in vivo gene therapy using onco-retroviral or lentiviral vectors often led to long-term FVIII or FIX expression from transduced hepatocytes, ex vivo approaches were generally hampered by either low or transient expression of FVIII or FIX levels in vivo and/or inefficient engraftment. Furthermore, immune responses against the transgene product remain a major issue that must be resolved before the full potential of these vectors eventually can be exploited clinically. Nevertheless, the continued progress in vector design combined with a better understanding of vector biology may ultimately yield more effective gene therapy approaches using these integrating vectors.