Non-integrating Lentiviral Research Articles

Gene therapy encoding cell cycle factors to treat chronic ischemic heart failure in rats.

Gene therapies to induce cardiomyocyte (CM) cell cycle re-entry have shown a potential to treat subacute ischaemic heart failure (IHF) but have not been tested in the more relevant setting of chronic IHF. Our group recently showed that polycistronic non-integrating lentivirus encoding Cdk1/CyclinB1 and Cdk4/CyclinD1 (TNNT2-4Fpolycistronic-NIL) is effective in inducing CM cell cycle re-entry and ameliorating subacute IHF models and preventing the subsequent IHF-induced congestions in the liver, kidneys, and lungs in rats and pigs. Here, we aim to test the long-term efficacy of TNNT2-4Fpolycistronic-NIL in a rat model of chronic IHF, a setting that differs pathophysiologically from subacute IHF and has greater clinical relevance. Rats were subjected to a 2-h coronary occlusion followed by reperfusion; 4 weeks later, rats were injected intramyocardially with either TNNT2-4Fpolycistronic-NIL or LacZ-NIL. Four months post-viral injection, TNNT2-4Fpolycistronic-NIL-treated rats showed a significant reduction in scar size and a significant improvement in left ventricular (LV) systolic cardiac function but not in the LV dilatation associated with chronic IHF. A mitosis reporter system developed in our lab showed significant induction of CM mitotic activity in TNNT2-4Fpolycistronic-NIL-treated rats. This study demonstrates, for the first time, that TNNT2-4Fpolycistronic-NIL gene therapy induces CM cell cycle re-entry in chronic IHF and improves LV function, and that this salubrious effect is sustained for at least 4 months. Given the high prevalence of chronic IHF, these results have significant clinical implications for developing a novel treatment for this deadly disease.

Early protective effect of a ("pan") coronavirus vaccine (PanCoVac) in Roborovski dwarf hamsters after single-low dose intranasal administration.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the danger posed by human coronaviruses. Rapid emergence of immunoevasive variants and waning antiviral immunity decrease the effect of the currently available vaccines, which aim at induction of neutralizing antibodies. In contrast, T cells are marginally affected by antigen evolution although they represent the major mediators of virus control and vaccine protection against virus-induced disease. We generated a multi-epitope vaccine (PanCoVac) that encodes the conserved T cell epitopes from all structural proteins of coronaviruses. PanCoVac contains elements that facilitate efficient processing and presentation of PanCoVac-encoded T cell epitopes and can be uploaded to any available vaccine platform. For proof of principle, we cloned PanCoVac into a non-integrating lentivirus vector (NILV-PanCoVac). We chose Roborovski dwarf hamsters for a first step in evaluating PanCoVac in vivo. Unlike mice, they are naturally susceptible to SARS-CoV-2 infection. Moreover, Roborovski dwarf hamsters develop COVID-19-like disease after infection with SARS-CoV-2 enabling us to look at pathology and clinical symptoms. Using HLA-A*0201-restricted reporter T cells and U251 cells expressing a tagged version of PanCoVac, we confirmed in vitro that PanCoVac is processed and presented by HLA-A*0201. As mucosal immunity in the respiratory tract is crucial for protection against respiratory viruses such as SARS-CoV-2, we tested the protective effect of single-low dose of NILV-PanCoVac administered via the intranasal (i.n.) route in the Roborovski dwarf hamster model of COVID-19. After infection with ancestral SARS-CoV-2, animals immunized with a single-low dose of NILV-PanCoVac i.n. did not show symptoms and had significantly decreased viral loads in the lung tissue. This protective effect was observed in the early phase (2 days post infection) after challenge and was not dependent on neutralizing antibodies. PanCoVac, a multi-epitope vaccine covering conserved T cell epitopes from all structural proteins of coronaviruses, might protect from severe disease caused by SARS-CoV-2 variants and future pathogenic coronaviruses. The use of (HLA-) humanized animal models will allow for further efficacy studies of PanCoVac-based vaccines in vivo.

Transient gene therapy using cell cycle factors reverses renin-angiotensin-aldosterone system activation in heart failure rat model.

The loss of cardiomyocytes after myocardial infarction (MI) leads to heart failure. Recently, we demonstrated that transient overexpression of 4 cell cycle factors (4F), using a polycistronic non-integrating lentivirus (TNNT2-4F-NIL) resulted in significant improvement in cardiac function in a rat model of MI. Yet, it is crucial to demonstrate the reversal of the heart failure-related pathophysiological manifestations, such as renin-angiotensin-aldosterone system activation (RAAS). To assess that, Fisher 344 rats were randomized to receive TNNT2-4F-NIL or control virus seven days after coronary occlusion for 2h followed by reperfusion. 4months after treatment, N-terminal pro-brain natriuretic peptide, plasma renin activity, and aldosterone levels returned to the normal levels in rats treated with TNNT2-4F-NIL but not in vehicle-treated rats. Furthermore, the TNNT2-4F-NIL-treated group showed significantly less liver and kidney congestion than vehicle-treated rats. Thus, we conclude that in rat models of MI, TNNT2-4F-NIL reverses RAAS activation and subsequent systemic congestion.

Transient Cell Cycle Induction in Cardiomyocytes to Treat Subacute Ischemic Heart Failure.

The regenerative capacity of the heart after myocardial infarction is limited. Our previous study showed that ectopic introduction of 4 cell cycle factors (4F; CDK1 [cyclin-dependent kinase 1], CDK4 [cyclin-dependent kinase 4], CCNB [cyclin B1], and CCND [cyclin D1]) promotes cardiomyocyte proliferation in 15% to 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction in mice. Using temporal single-cell RNA sequencing, we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Using rat and pig models of ischemic heart failure, we aimed to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure. Temporal bulk and single-cell RNA sequencing and further biochemical validations of mature human induced pluripotent stem cell-derived cardiomyocytes treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 hours after infection with 4F, which was associated mainly with sarcomere disassembly and metabolic reprogramming (n=3/time point/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic nonintegrating lentivirus (NIL) encoding 4F; each is driven by a TNNT2 (cardiac troponin T isoform 2) promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially 1 week after myocardial infarction in rats (n=10/group) or pigs (n=6-7/group). Four weeks after injection, TNNT2-4Fpolycistronic-NIL-treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus-treated animals. At 4 months after treatment, rats that received TNNT2-4Fpolycistronic-NIL still showed a sustained improvement in cardiac function and no obvious development of cardiac arrhythmias or systemic tumorigenesis (n=10/group). This study provides mechanistic insights into the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors owing to the use of a novel transient and cardiomyocyte-specific viral construct.

Abstract 10492: Preclinical Evaluation of Transient and Cardiomyocyte Specific Gene Therapy for the Treatment of Subacute Ischemic Heart Failure

Rationale: Treatment of heart failure is limited due to the limited regenerative capacity of cardiomyocytes. Our group has shown that ectopic expression of Cdk1/CyclinB1 and Cdk4/CyclinD1 (named 4F) via adenovirus vector promotes cardiomyocyte proliferation in 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction (MI). Objective: To introduce 4F specifically and transiently into cardiomyocytes to induce selective cardiomyocyte regeneration and limit any tumorigenic potential in other organs. Methods and Results: A polycistronic non-integrating lentivirus encoding 4F, each driven by a TNNT2 promoter (TNNT2-4F-NIL), was generated to induce transient and specific expression of 4F cardiomyocytes. TNNT2-4F-NIL promotes proliferation of 15% of cardiomyocytes in vivo after MI as assessed in lineage-tracing mice (α-MHC-Cre-MADM). Rats and pigs were subjected to coronary occlusion/reperfusion to induce MI. One week later, animals were injected (intramyocardially) with either TNNT2-4F-NIL or control-NIL. Four weeks after injection, TNNT2-4F-NIL treated rats and pigs showed a significant improvement in left ventricular ejection fraction (EF) compared to controls (TNNT2-4F-NIL rats 54.3±1.78% vs control-NIL 42.97±2.60%; n=9/group, p =0.003, TNNT2-4F-NIL pigs 37±2.3% vs control-NIL 28.4±1.2%; n=6/group, p =0.01). Histological analyses showed a 25-30% reduction in scar size in both rats and pigs treated with TNNT2-4F-NIL vs. control-NIL (p<0.05). To assess the long-term efficacy and safety of TNNT2-4F-NIL, a 4-months follow-up experiment after MI and virus treatment was performed during which cardiac function was assessed monthly. TNNT2-4F-NIL treated rats showed sustained improvement in cardiac function (EF in TNNT2-4F-NIL 48.4±3% vs in control-NIL 33.6±3%; n=6/group, p =0.008). There was no obvious development of arrhythmias and no evidence of oncogenicity or toxicity in other organs in TNNT2-4F-NIL treated rats or in the control group. Conclusion: This study provides a novel approach to treat heart failure, with the use of transient and cardiomyocyte-specific gene therapy that promotes long-term functional improvement after a single treatment with no obvious adverse effect.

Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

BackgroundGene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone.MethodsA two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein.ResultsTransduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir.ConclusionsOur study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

Abstract 14391: Transient Cell Cycle Induction in Cardiomyocytes is a Promising Approach to Treat Ischemia Induced Heart Failure

Rationale: The regenerative capacity of the heart to repair itself after myocardial infarction (MI)is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 andCdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in vitro and in vivo andimproves cardiac function after MI. However, its clinical application is limited due to the concernsfor tumorigenic potential in other organs. Objectives: To first, identify on a single cell transcriptomic basis the necessary reprogrammingsteps that cardiomyocytes need to undertake to progress through the proliferation processfollowing 4F overexpression, and then, to determine the pre-clinical efficacy of transient andcardiomyocyte specific expression of 4F in improving cardiac function after MI in small and largeanimals. Methods and Results: Temporal bulk and single cell RNAseq of mature hiPS-CMs treated with4F or LacZ control for 24, 48, or 72 h revealed full cell cycle reprogramming in 15% of thecardiomyocyte population which was associated with sarcomere disassembly and metabolicreprogramming. Transient overexpression of 4F specifically in cardiomyocytes was achievedusing non-integrating lentivirus (NIL) driven by TNNT2 (TNNT2-4F-NIL). One week after inductionof ischemia-reperfusion injury in rats or pigs, TNNT2-4F-NIL or control virus was injectedintramyocardially. Compared with controls, rats or pigs treated with TNNT2-4F-NIL showed a 20-30% significant improvement in ejection fraction and scar size four weeks after treatment, asassessed by echocardiography and histological analysis. Quantification of cardiomyocyteproliferation in pigs using a novel cytokinesis reporter showed that ~10% of the cardiomyocyteswithin the injection site were labelled as daughter cells following injection with TNNT2-4F-NILcompared with ~0.5% background labelling in control groups. Conclusions: We provide the first understanding of the process of forced cardiomyocyteproliferation and advanced the clinical applicability of this approach through minimization ofoncogenic potential of the cell cycle factors using a novel transient and cardiomyocyte-specificviral construct.

Episomal lentiviral vectors confer erythropoietin expression in dividing cells

Lentiviral vectors are now widely considered as one of the most common gene delivery tools for dividing and non-dividing cells. However, insertional mutagenesis has been found in clinical trials with retroviral vectors, which poses a safety risk. The use of non-integrating lentiviral (NIL) vectors, which avoid integration, eliminates the insertional mutagenesis problem. These NIL vectors are unable to mediate stable gene delivery into dividing cells, which makes them of limited use in the clinical practice of gene therapy. In this study, we constructed a NIL vector which harbors the scaffold/matrix attachment region (S/MAR) sequence and a therapeutic gene. NIL retained episomal erythropoietin (EPO) gene expression for 74days in dividing cells both with and without selection. Furthermore, Southern blot analysis showed that the NIL vector was retained extrachromosomally in CHO cells. In conclusion, the NIL vector based on an S/MAR sequence retained the extrachromosomal expression of a therapeutic gene in dividing cells. Our results show that NIL vectors maybe a safe and effective means of gene delivery, which is of potential clinical significance.

Anchored Lentiviral Vector Episomes for Stem Cell Gene Therapy in Fanconi Anemia.

Fanconi anemia (FA) is an autosomal recessive, multisystem DNA repair disorder with prominent defects in the hematopoietic stem cell maintenance that result in the progressive attrition and failure in the early school age. Allogeneic stem cell transplantation has proved curative for patients with suitable donors. This, along with the characteristic survival advantage of phenotypically normal over non-corrected FA stem cells underscores the compelling rationale for stem cell gene therapy in the FA. While integrating lentiviral vectors (LV) have become the preferred platform for genetic correction in several hematologic and immunodeficiency disorders, the residual oncogenic potential by these vectors raises concerns in the FA stem cells about insertional mutagenic genetic lesions. On this backdrop, investigators are developing a new generation of non-integrating viral vectors capable of nuclear persistence through serial mitotic cycles and stable under selection to offset the comparatively lower transduction rates. Here, we review the competing approaches to develop such non-integrating lentiviral (NILV) episome vectors that faithfully replicate in the stem cells.

Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells.

Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal genetransfer.

Chimeric antigen receptor (CAR) T‐cell therapy is a new successful treatment for refractory B‐cell leukemia. Successful therapeutic outcome depends on long‐term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long‐term cell engineering method using non‐integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV‐S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV‐S/MAR‐engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19+ target cell recognition as LV‐engineered T cells and are as effective in controlling tumor growth in vivo. We propose that NILV‐S/MAR vectors are superior to current options as they enable long‐term transgene expression without the risk of insertional mutagenesis and genotoxicity.

Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification.

Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy.

Abstract A171: Long-term episomal gene transfer for safe engineering of T cells for adoptive cell therapy of cancer

Abstract Success in T-cell therapy of cancer is currently relying on long-term gene expression owing to retrovirus/lentivirus (RV/LV) integration. However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal and long-term cell engineering method using non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element for either over-expression or down-regulation of genes. T-cells engineered with a NILV-S/MAR(CD19CAR) vector have similar levels of CAR expression as T-cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV-S/MAR-engineered CD19CAR-T-cells exhibited similar cytotoxic capacity upon CD19+ target cells recognition as LV-engineered T-cells. We propose that NILV-S/MAR vectors are superior to current options for enabling long-term gene expression without the risk of insertional mutagenesis. Citation Format: Chuan Jin, Mohanraj Ramachandran, Grammatiki Fotaki, Berith Nilsson, Magnus Essand, Di Yu. Long-term episomal gene transfer for safe engineering of T cells for adoptive cell therapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A171.

Antiangiogenic Eye Gene Therapy.

The idea of treating disease in humans with genetic material was conceived over two decades ago and with that a promising journey involving development and efficacy studies in cells and animals of a large number of novel therapeutic reagents unfolded. In the footsteps of this process, successful gene therapy treatment of genetic conditions in humans has shown clear signs of efficacy. Notably, significant advancements using gene supplementation and silencing strategies have been made in the field of ocular gene therapy, thereby pinpointing ocular gene therapy as one of the compelling "actors" bringing gene therapy to the clinic. Most of all, this success has been facilitated because of (1) the fact that the eye is an effortlessly accessible, exceedingly compartmentalized, and immune-privileged organ offering a unique advantage as a gene therapy target, and (2) significant progress toward efficient, sustained transduction of cells within the retina having been achieved using nonintegrating vectors based on recombinant adeno-associated virus and nonintegrating lentivirus vectors. The results from in vivo experiments and trials suggest that treatment of inherited retinal dystrophies, ocular angiogenesis, and inflammation with gene therapy can be both safe and effective. Here, the progress of ocular gene therapy is examined with special emphasis on the potential use of RNAi- and protein-based antiangiogenic gene therapy to treat exudative age-related macular degeneration.

93. Targeting Resting T Cells with Non-Integrating Lentivirus Encoding CCR5-ZFN Suppresses HIV-1 Infection In Vitro and in Humanized Mice

93. Targeting Resting T Cells with Non-Integrating Lentivirus Encoding CCR5-ZFN Suppresses HIV-1 Infection In Vitro and in Humanized Mice

CCR5 Gene Editing of Resting CD4(+) T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice.

CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4+ T cells. Both activated and resting primary CD4+ T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4+ T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2rγc null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN–transduced resting CD4+ T cells from treatment naïve as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy.

Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors

Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using non-integrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.

Transient Gene Expression by Nonintegrating Lentiviral Vectors

Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34+ hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (∼90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 104-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.

391. Effective Gene Therapy with Non-Integrating Lentivirus Vectors

Gene therapy strategies need to achieve efficient transgene expression whilst minimizing toxic effects. Retrovirus vectors are very effective in several systems, but they have been shown to cause malignancies through insertional mutagenesis, one of the most serious side-effects identified to date in pre-clinical studies and clinical trials. The risk of insertional mutagenesis has been considered unavoidable because no gene expression has been demonstrated in vivo in the absence of retroviral integration. However, retrovirus and lentivirus vectors can be rendered integration-defective by mutation in the integrase coding sequence. Following reverse transcription, mutant vectors fail to integrate and instead accumulate in transduced cells as double-stranded DNA circles. These circular forms are non-replicating but very stable and will persist if not diluted by cell division. Contrary to the results from previous in vivo studies, recent reports have shown efficient transgene expression in vitro from integration-deficient lentivirus vectors in post-mitotic and growth-arrested cells. These vectors also led to transient transgene expression or, in combination with episomal systems, to stable expression in proliferating cells in vitro. Given these developments we decided to re-evaluate the proficiency of integration-defective lentiviral vectors for in vivo expression in rodents. We now show that integration-deficient HIV-1 vectors lead to highly efficient, long-term marker gene expression in post-mitotic ocular and brain tissues. We also demonstrate efficient rescue in two rodent models of retinal degeneration, the Rpe65rd12/rd12 mouse and the Mertk-deficient Royal College of Surgeons (RCS) rat. The relevance of these studies for clinical application is highlighted by the fact that we have been able to transduce excised human retinal pigment epithelium with integration-deficient vectors. In conclusion, we demonstrate that integration-deficient HIV-1 vectors transduce post-mitotic tissues in vivo as efficiently as their integrating counterparts. For clinical applications in quiescent tissues these non-integrating vectors should virtually abolish the risk of insertional mutagenesis-induced malignancy.

416. Transient Gene Expression by Non-Integrating Lentiviral (NIL) Vectors

The ability to express genes transiently in stem cells would have a wide range of useful applications; for example the introduction of genes that could stimulate cell division, induce commitment to pathways of differentiation, or provide transient resistance to chemotherapeutic agents. Despite the potential usefulness of transient gene expression in hematopoietic stem cells (HSCs), there are still few methods that achieve gene delivery to these cells at high levels. Vectors based on attenuated Retroviruses and Lentiviruses have been used to efficiently and stably introduce genes into cells for long term expression. Their ability to stably transduce cells relies on the fact that their genome encodes an integrase protein that specifically recognizes sequences in the viral long terminal repeats (LTRs) and catalyses their insertion into host chromosomes. Due to their high efficiency of gene delivery and relatively low toxicity, we hypothesize that Non-Integrating Lentiviral (NIL) vectors may also be extremely useful in situations where transient gene expression is required. NIL vectors are lentiviral vectors harboring combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences located in the viral LTRs. Therefore, NIL vectors are designed to disable integration without affecting entry into cells, thus promoting transient gene expression. We have used these NIL vectors to transduce both HSCs and cell lines in order to assess their efficacy, as measured through transient eGFP expression. Our data demonstrate that transductions using the most effective NIL vectors harboring both LTR and integrase mutations resulted in initial high level transient eGFP expression (~90%), diminishing towards background in <1 month. This is in contrast to the wild-type vector which causes continued expression of eGFP (up to 3 months in cell lines). Encouragingly, NIL vectors performed equally well in primary human CD34+ cells as they did in cell lines. Southern blot analyses of transduced Jurkat cells depicted the loss of detectable NIL vector sequence after 1 month. Additionally the Southern analyses revealed important information regarding the frequency of formation of 1 or 2 LTR circles in comparison to linear DNA, which are typical molecular species formed by such non-integrating viral vectors. Through PCR and sequencing we have studied the number and nature of integrations of the NIL vectors in pools and colonies of Jurkat cells and compared this information to results from Jurkat cells exposed to the wild-type lentiviral vector. We believe that these studies may lead to the development of a vector system capable of highly efficient, but transient gene expression in human stem and mature cells.