LV-mediated Gene Transfer Research Articles

Breaching the Delivery Barrier: Chemical and Physical Airway Epithelium Disruption Strategies for Enhancing Lentiviral-Mediated Gene Therapy

The lungs have evolved complex physical, biological and immunological defences to prevent foreign material from entering the airway epithelial cells. These mechanisms can also affect both viral and non-viral gene transfer agents, and significantly diminish the effectiveness of airway gene-addition therapies. One strategy to overcome the physical barrier properties of the airway is to transiently disturb the integrity of the epithelium prior to delivery of the gene transfer vector. In this study, chemical (lysophosphatidylcholine, LPC) and physical epithelium disruption using wire abrasion were compared for their ability to improve airway-based lentiviral (LV) vector mediated transduction and reporter gene expression in rats. When luciferase expression was assessed at 1-week post LV delivery, LPC airway conditioning significantly enhanced gene expression levels in rat lungs, while a long-term assessment in a separate cohort of rats at 12 months revealed that LPC conditioning did not improve gene expression longevity. In rats receiving physical perturbation to the trachea prior to gene delivery, significantly higher LacZ gene expression levels were found when compared to LPC-conditioned or LV-only control rats when evaluated 1-week post gene transfer. This proof-of-principle study has shown that airway epithelial disruption strategies based on physical perturbation substantially enhanced LV-mediated airway gene transfer in the trachea.

Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers.

Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

Preclinical Testing of the Safety and Tolerability of Lentiviral Vector-Mediated Above-Normal Alpha-L-Iduronidase Expression in Murine and Human Hematopoietic Cells Using Toxicology and Biodistribution Good Laboratory Practice Studies.

In order to support the clinical application of hematopoietic stem cell (HSC) gene therapy for mucopolysaccharidosis I (MPS I), biosafety studies were conducted to assess the toxicity and tumorigenic potential, as well as the biodistribution of HSCs and progenitor cells (HSPCs) transduced with lentiviral vectors (LV) encoding the cDNA of the alpha-iduronidase (IDUA) gene, which is mutated in MPS I patients. To this goal, toxicology and biodistribution studies were conducted, employing Good Laboratory Practice principles. Vector integration site (IS) studies were applied in order to predict adverse consequences of vector gene transfer and to obtain HSC-related information. Overall, the results obtained in these studies provided robust evidence to support the safety and tolerability of high-efficiency LV-mediated gene transfer and above-normal IDUA enzyme expression in both murine and human HSPCs and their in vivo progeny. Taken together, these investigations provide essential safety data to support clinical testing of HSC gene therapy in MPS I patients. These studies also underline criticisms associated with the use of currently available models, and highlight the value of surrogate markers of tumorigenicity that may be further explored in the future. Notably, biological evidence supporting the efficacy of gene therapy on MPS I disease and its feasibility on patients' HSCs were also generated, employing clinical-grade LVs. Finally, the clonal contribution of LV-transduced HSPCs to hematopoiesis along serial transplantation was quantified in a minimum of 200-300 clones, with the different level of repopulating cells in primary recipients being reflected in the secondary.

Induction of Hematopoietic Microchimerism by Gene-Modified BMT Elicits Antigen-Specific B and T Cell Unresponsiveness toward Gene Therapy Products

Gene therapy is a promising treatment option for hemophilia and other protein deficiencies. However, immune responses against the transgene product represent an obstacle to safe and effective gene therapy, urging for the implementation of tolerization strategies. Induction of a hematopoietic chimerism via bone marrow transplantation (BMT) is a potent means for inducing immunological tolerance in solid organ transplantation. We reasoned, here, that the same viral vector could be used, first, to transduce BM cells for inducing chimerism-associated transgene-specific immune tolerance and, second, for correcting protein deficiencies by vector-mediated systemic production of the deficient coagulation factor. Evaluation of strategies to induce B and T cell tolerance was performed using ex vivo gene transfer with lentiviral (LV) vectors encoding coagulation factor IX (FIX) or the SIINFEKL epitope of ovalbumin. Following induction of microchimerism via BMT, animals were challenged with in vivo gene transfer with LV vectors. The experimental approach prevented humoral immune response against FIX, resulting in persistence of therapeutic levels of circulating FIX, after LV-mediated gene transfer in vivo. In an ovalbumin model, we also demonstrated that this approach effectively tolerized the CD8+ T cell compartment in an antigen-specific manner. These results provide the proof-of-concept that inducing a microchimerism by gene-modified BMT is a powerful tool to provide transgene-specific B and T cell tolerance in a gene therapy setting.

Perforin Gene Transfer Into Hematopoietic Stem Cells Improves Immune Dysregulation in Murine Models of Perforin Deficiency

Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8+ T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8+ lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL.

T-Cell-Mediated Suppression: From Bench to Bedside

T-Cell-Mediated Suppression: From Bench to Bedside

A TLR and Non-TLR Mediated Innate Response to Lentiviruses Restricts Hepatocyte Entry and Can be Ameliorated by Pharmacological Blockade

Lentiviral vector (LV)-mediated gene transfer is a promising method of gene therapy. We previously reported that systemic injection of HIV-based LV triggers a transient inflammatory response. Here, we carried out studies to better characterize this response, and to develop a strategy to overcome the adverse effects of interferon (IFN) on LV-mediated gene transfer. We profiled gene expression in the liver after LV administration using deep-sequencing (RNA-seq), and identified several innate response pathways. We examined the response to LV in MyD88-TRIF knockout mice, which are incapable of toll-like receptor (TLR) signaling. Unexpectedly, the IFN response to LV was not reduced in the liver indicating that a non-TLR pathway can recognize LV in this organ. Indeed, blocking reverse transcription with azidothymidine (AZT) reduced the IFN response only in the liver, suggesting that proviral DNA can be a trigger. To block the inflammatory response, we pretreated mice with a short course of dexamethasone (Dex). At 4 hours post-treatment, all the IFN-induced genes were normalized. By blocking the inflammatory response, hepatocyte transduction was dramatically increased, which in turn doubled the level of human factor IX (FIX) produced by a hepatocyte-specific LV. Our studies uncover new insights into LV-induced immune responses in the liver, and provide a means to increase the safety and efficiency of LV-mediated gene transfer.

Generation of stable, high‐producing cho cell lines by lentiviral vector‐mediated gene transfer in serum‐free suspension culture

Lentivirus-derived vectors (LVs) were studied for the generation of stable recombinant Chinese hamster ovary (CHO) cell lines. Stable pools and clones expressing the enhanced green fluorescent protein (eGFP) were selected via fluorescence-activated cell sorting (FACS). For comparison, cell pools and cell lines were also generated by transfection, using the LV transfer plasmid alone. The level and stability of eGFP expression was greater in LV-transduced cell lines and pools than in those established by transfection. CHO cells were also infected at two different multiplicities of infection with an LV co-expressing eGFP and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). At 2-day post-infection, clonal cell lines with high eGFP-specific fluorescence were recovered by FACS. These clones co-expressed TNFR:Fc with yields of 50-250 mg/L in 4-day cultures. The recovered cell lines maintained stable expression over 3 months in serum-free suspension culture without selection. In conclusion, LV-mediated gene transfer provided an efficient alternative to plasmid transfection for the generation of stable and high-producing recombinant cell lines.

Promoter-specific lentivectors for long-term, cardiac-directed therapy of Fabry disease

In Fabry disease a deficiency of α-galactosidase A (α-gal A) activity leads to accumulation of globotriaosylceramide (Gb3) in various tissues including the heart. A specific cardiac variant of Fabry disease has also been described. Previously we have demonstrated the feasibility of gene therapy for Fabry disease. Here, to provide efficient transfer and increased specificity of transgene expression, we synthesized lentiviral vectors (LVs) with myocardial-specific promoters including: α-myosin heavy chain (α-MHC), myosin light chain (MLC2v), and cardiac troponin T (cTnT). Initially, neonatal Balb/c mice were injected with such LV constructs engineering expression of luciferase. One month post-injection, we found specific expression of luciferase in hearts of recipient animals when compared with transgene expression driven by the standard EF1-α promoter. To examine the feasibility of long-term therapy specifically targeting the heart, recombinant LV/α-gal A therapeutic vectors with analogous cardiac promoters were generated and injected into numerous neonatal Fabry mice. No immune response against the corrective α-gal A hydrolase was observed in the treated mice. Serum α-gal A activity of 10-week-old Fabry mice was increased in LV/α-gal A-injected animals compared to controls. In 28-week-old Fabry mice we observed significantly decreased Gb3 accumulation. Neonatal injections with LVs harboring cardiac-specific promoters may thus be an effective long-term treatment strategy for heart manifestations and cardiac variant Fabry disease. These results can be also extended to other progressive pathologies of the heart.

Effective in vivo and ex vivogene transfer to intestinal mucosa by VSV-G-pseudotyped lentiviral vectors

BackgroundGene transfer to the gastrointestinal (GI) mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD), GI infections, and cancer.MethodsWe evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G)-pseudotyped lentiviral vectors (LV) for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal) or firefly-luciferase (LV-fLuc) reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression and identity of transduced cell types were examined by histochemical and immunofluorescence staining.ResultsImaging studies showed positive fLuc signals in murine distal colon; β-Gal-positive cells were found in both murine and human intestinal tissue. In the murine model, β-Gal-positive epithelial and lamina propria cells were found to express cytokeratin, CD45, and CD4. LV-transduced β-Gal-positive cells were also seen in human colorectal explants, consisting mainly of CD45, CD4, and CD11c-positive cells confined to the LP.ConclusionsWe have demonstrated the feasibility of LV-mediated gene transfer into colonic mucosa. We also identified differential patterns of mucosal gene transfer dependent on whether murine or human tissue was used. Within the limitations of the study, the LV did not appear to induce mucosal damage and were not distributed beyond the distal colon.

Real-time in vivo bioluminescence imaging of lentiviral vector–mediated gene transfer in mouse testis

Although much research has focused on transferring exogenous genes into living mouse testis to investigate specific gene functions in spermatogenic, Sertoli, and Leydig cells, relatively little is known regarding real-time gene expression in vivo. In this study, we constructed a bicistronic lentiviral vector (LV) encoding firefly luciferase and enhanced green fluorescence protein (EGFP); this was a highly efficient in vivo gene transfer tool. After microinjecting LV into the seminiferous tubules the ICR mouse testis, we detected luciferase and EGFP expression in vivo and ex vivo in the injected tubules using bioluminescence imaging (BLI) with the IVIS-200 system and fibered confocal fluorescence microscopy (CellViZio), respectively. In addition, with an in vivo BLI system, luciferase expression in the testis was detected for ∼3 mo. Furthermore, EGFP expression in seminiferous tubules was confirmed in excised testes via three-dimensional fluorescent imaging with a confocal laser-scanning microscope. With immunostaining, EGFP expression was confirmed in several male germ cell types in the seminiferous tubules, as well as in Sertoli and Leydig cells. In conclusion, we demonstrated that real-time in vivo BLI analysis can be used to noninvasively (in vivo) monitor long-term luciferase expression in mouse testis, and we verified that EGFP expression is localized in seminiferous tubules after bicistronic LV-mediated gene transfer into mouse testes. Furthermore, we anticipate the future use of in vivo BLI technology for real-time study of specific genes involved in spermatogenesis.

A lentiviral vector‐based adenovirus fiber‐pseudotyping approach for expedited functional assessment of candidate retargeted fibers

Many studies aimed at retargeting adenovirus (Ad) rationally focus on genetic modification of fiber, which is the primary receptor-binding protein of Ad. Retargeted fibers ultimately require functional validation in the viral context. Lentiviral vectors (LV) were used to express fiber variants in cells. Infections with a fiber gene-deleted Ad vector yielded fiber-pseudotyped viruses. An enzyme-linked immunosorbent assay and slot blot-based assays probed target binding-ability of retargeted fibers. Differential treatments with an alkylating agent prior to western blot analysis allowed for examination of intra- and extracellular redox states of fibers. In the present study, LV-based fiber-pseudotyping of Ad is presented as an accelerated means to test new fibers. LV-mediated gene transfer yielded stable and uniform populations of fiber variant-expressing cells. These populations were found to effectively support fiber-pseudotyping of Ad. As a secondary objective of the study, we functionally assessed a chimeric fiber harboring a tumor antigen-directed single-chain antibody fragment (scFv). This fiber was shown to trimerize and achieve a degree of binding to its antigenic target. However, its capsid incorporation ability was impaired and, moreover, it was unable to confer a detectable level of target binding upon Ad. Importantly, subsequent analyses of this fiber revealed the improper folding of its scFv constituent. LV-based fiber-pseudotyping was established as a convenient method for testing modified fibers for functionality within Ad particles. Furthermore, a new chimeric fiber was found to be inadequate for Ad retargeting. The folding difficulties encountered for this particular fiber might be generally inherent to the use (i.e. for genetic Ad capsid incorporation) of complex, disulfide bridge-containing natural ligands.

Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)–deficient mice and corrects their immune and metabolic defects

AbstractAdenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA–/– mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.

Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation

Although Pdx1-VP16 expression induces hepatic cell transdifferentiation into pancreatic precursor cells (WB-1), these incompletely reprogrammed cells fail to become glucose-sensitive insulin-producing cells in the absence of the activation of late-stage pancreatic transcription factors. As Pax4 promotes late-stage β-cell differentiation and maturation, we generated lentiviral vector (LV) containing mouse Pax4 gene and developed two hepatic cell lines expressing Pax4 in the absence (WB-2 cells) or presence (WB-1A cells) of Pdx1-VP16, via LV-mediated gene transfer. Functional Pax4 protein expression in WB-2 and WB-1A cells was confirmed by electrophoretic mobility shift assay and Pdx1-VP16 protein expression in WB-1 and WB-1A cells was confirmed by Western blotting. Activation of Pax4 resulted in the expression of the late-stage transcription factors, including Pax6, Isl-1, and MafA, and generated a gene expression profile for WB-1A cells similar to that of functional rat insulinoma INS-1 cells. Insulin abundance in WB-1A cells was demonstrated by immunostaining. WB-1A cells exhibited glucose-responsive insulin release in vitro, and caused a rapid reversal of hyperglycemia following cell transplantation into streptozotocin-induced diabetic mice. Intraperitoneal glucose tolerance test showed a normal glucose response in WB-1, and WB-1A transplanted mice similar to that of normal mice. Removal of transplanted WB-1A cells resulted in a return of hyperglycemia, confirming that they were responsible for the observed normoglycemia. The explanted WB-1A cells exhibited strong insulin staining comparable to native islet β-cells. These studies indicate that activation of Pax4 in Pdx1-VP16-expressing cells reprograms pancreatic precursor-like WB-1 cells into glucose-responsive, more mature insulin-producing cells.

Lentiviral-Mediated Fetal Gene Therapy for Monogenic Disorders: Development of an in vitro Rabbit Model

Objective: Fetal gene replacement is a novel, potential therapy for monogenic disorders which are diagnosed prenatally. The purpose of this study was to develop in vitro, respiratory-epithelium targeted, lentiviral (LV)-mediated gene transfer in fetal rabbit tracheas. Methods: Via triple plasmid transfection, vesicular stomatitis virus-G (VSV-G)-pseudotyped LV vector containing green fluorescent protein (GFP) marker gene, under the control of a cytomegalovirus promoter was constructed. LV bioavailability in rabbit amniotic fluid (AF) was evaluated by infectivity assays of 293T cell monolayers in variable concentrations of AF. Fetal tracheas from time-mated rabbits (term gestation, G = 31 days) were collected on G 23–25 days, and placed in tissue culture (substrate-enriched DMEM, 37°C, 5% CO₂/room air). The tracheal cultures were transfected with 1 × 10⁵ LV particles, and analyzed daily for: reporter gene by polymerase chain reaction, and reporter gene product (GFP) by whole-mount fluoroscopy and immunohistochemistry. Results: 293T cell infectivity assays confirmed bioavailability of LV in rabbit AF. Following in vitro transfection, GFP DNA and GFP were detectable in fetal rabbit tracheas by 4 and 5 days, respectively. Immunocytochemistry localized GFP to the luminal aspect of tracheal epithelium. Conclusions: In vitro, LV-mediated GFP gene transfer to fetal rabbit tracheas occurs within 4 days, and gene expression is evident by 5 days post-transfection. This observation, and the bioavailability of LV through AF, suggests the appropriateness of this model for the future evaluation of in vivo, transamniotic gene delivery strategies.

358. Gene Transfer to the Airway Epithelium Mediated by a Third Generation HIV-1 Based Vector: Efficiency and Role of Heparan Sulfate

HIV-1 derived lentiviral (LV) vectors seem to be promising vehicles for gene transfer to the airway epithelium since they integrate in the host genome of non-dividing cells ensuring long-term expression of the transgene. Previous studies have shown that LV vectors could transduce the airway epithelium in vivo from the apical surface, only when tight junctions are disrupted or LV are pseudotyped with heterologous envelopes other than glycoprotein G of the vesicular stomatitis virus (VSV-G). Our study was aimed to investigate the efficiency of an improved VSV-G pseudotyped lentiviral vector, which contains the central polypurine tract (cPPT) and the Woodchuck post-translational regulatory elements, in human respiratory cells and in the murine lung. The vector PPT-GFP was injected intratracheally in C57BL/6 mice at the dose of 4.5|[times]|10E6 TU. After 48 hours, lungs were fixed in paraformaldehyde and cryosections stained with an antibody against GFP. Results show that PPT-GFP transduced the murine airway epithelium in the bronchial and alveolar compartments. Transgene expression was observed also after two weeks of infection with a LV dose of 1|[times]|10E8 TU. Hystological analysis showed no evidence of tissue damage in the lung, suggesting that this LV vector is not cytotoxic. Since heparan sulfate (HS) has been described to mediate infection by VSV-G-pseudotyped retroviral vectors and HIV-1, the expression of this glycosaminoglycan by human respiratory cells and its involvement in LV-mediated gene transfer were studied. Human bronchial epithelial cells (16HBE) and CF tracheal epithelial cells (CFT1) were analyzed for HS expression by flow cytometry and confocal microscopy. HS was expressed by 85-88% of cells, the expression being much stronger on the basolateral side in polarized epithelial cells. To correlate the efficiency of LV mediated transduction with HS expression, polarized CFT1 cells were infected with the lentivirus and some samples were pre-incubated with EGTA (Ethylene glycol-O,O-bis-[2-amino-ethyl]-N,N,N,N,-tetraacetic acid), which disrupts the epithelial tight junctions and allows the LV particles to access the basolateral side of the epithelium. Analysis of GFP expression by FACS indicates that the pre-conditioning increased the transduction efficiency, being the percentage of positive cells of 55%, when the infection was performed without pretreatment, and 71% when the cells were incubated with EGTA. To evaluate the interaction between glycosaminoglycans expressed on the apical surface and lentiviral particles, the LV vector was pre-incubated with heparin and then incubated with 16HBE cells. Heparin determined a dose-dependent inhibition of transduction efficiency up to 65% of untreated vector. Our study shows that 1) The PPT-GFP vector transduce murine airways and human polarized cells, without requirement of disruption of the epithelial barrier integrity and without cytotoxicy 2) HS might be a receptor involved in VSV-G-pseudotyped LV-mediated gene transfer.

In Vivo Bone Marrow Stem Cell Gene Transfer in Mice by In Situ Delivery of a 3rd-Generation Lentiviral Vector Using Intrafemoral Injection.

HIV-based lentiviral vectors (LV) have become powerful tools for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer by “in situ” bone cavity injection has not been well unexplored. This in vivo approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if intra bone marrow (iBM) injection of LV can efficiently transduce BM stem cells, a 3rd-generation self-inactivating LV, containing eGFP regulated transcriptionally by a CMV promoter, was used to inject intra-femorally into adult Boy/J Ly45.1 mice (at 2 x106 IU/mouse). Blood counts were normal in all LV-injected mice (n=4) and buffer-injected mice (n=4). GFP-expressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. To evaluate gene transfer and transgene expression in multi-potential progenitors, the colony-forming cell (CFC) assay was performed using low-density bone marrow 4-months post injection, resulting in 4–5% GFP+ CFU-granulocyte macrophage (CFU-GM) and CFU-granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM). This was consistent with the 4-color FACS analysis data demonstrating that up to 3.7% of HSC/progenitors (Lin−c-kit+Sca1+ cells) from LV-injected mice expressed GFP transgene 4-months post injection. To further evaluate HSC gene transfer, BM from injected mice was transplanted into lethally irradiated adult C57/Bl6 (Ly45.2) mice. Four months later, successful engraftment was demonstrated in all BM recipients with 97–99% donor-derived cells in PBL and 91–93.3% in BM. Moreover, significant levels of GFP+ CFU were observed in all recipients ranging from 8.4 ± 2.3% to 17.7 ± 4.2%. To further study the LV-mediated gene transfer profile in HSC, mesenchymal progenitors and systemic distribution by intra-femoral injection, molecular analyses are in progress including real-time QPCR and clonality analysis by LAM-PCR. Our results indicate successful LV-mediated gene transfer into HSC by iBM injection. These data would potentially open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.

661. Immune System Regulation of Transgene Expression in the Brain 3: Effects of the Immune Response on Transgene Expression from HIV-Derived Lentiviral Vectors Injected into the Rat Striatum

HIV-derived pseudotyped lentiviral vectors are potent vectors for the transfer of potentially therapeutic genes into the brain. However, the efficiency of LV-mediated gene transfer into the brain has not been examined in detail. Further, nothing is known on the effects of the systemic priming of an anti-lentiviral immune response on transgene expression and inflammation in the brain. The self-inactivating pRRLsin.cPPT.CMV.Wpre, and pRRLsin.cPPT.ALB.Wpre constructs were used to generate VSV-pseudotyped LV stocks expressing either GFP (LV-GFP) or human FIX (LV-hFIX). Titers for LV-GFP were determined on HeLa and mouse liver (MLP29) cells and were approx. 5 × 109 to 2 × 1010 transducing units (TU)/ml with 100–400 ug HIV-1 p24/ml. To study the innate inflammatory responses to LV vectors in the CNS, LV-GFP was injected into the striatum at doses of 1 × 102−1 × 107 TU. Transduced cells could be found at all doses, and transgene expression increased up to the highest dose. Even at the highest dose, there was no statistically significant increase in the influx of either activated macrophages or CD8+-T cells. To analyze the effects of the systemic anti-LV immunization on gene expression in the CNS, LV-GFP was injected into the striatum, followed 30 days later, by the systemic immunization with either LV-GFP or LV-FIX, or the injection of saline. The effectiveness of the immune responses were determined by the presence of anti-envelope and anti-transgene antibodies by ELISA. In animals immunized against LV-FIX or injected with saline, there was no changes in the number of immunoreactive cells in the brain. However, in animals immunized against LV-GFP, there was a 30–50% reduction in the number of immunoreactive neurons. Concomitantly, there was a highly significant increase in the number of CD8+ T-cells in the brains of animals immunized with LV-GFP, but not those immunized with LV-FIX. This indicates that LV-injected into the brain cause minimal activation of the CNS innate inflammatory response. However, in the presence of a systemic anti-transgene immune response, activated lymphocytes can enter the brain and contribute to a down-regulation of LV-encoded transgene expression. Since LV-derived vectors do not express any vector proteins, immunization against a LV vector expressing a different transgene did not cause brain inflammation or down-regulation of CNS transgene expression. In conclusion, a systemic anti-transgene immune response can down-regulate gene expression from a LV vector in the CNS.

Late-Breaking Science

### H11 Kinase Promotes or Inhibits Cardiac Cell Growth and Survival, Depending on its Subcellular Distribution Christophe Depre, Anna Zajac, Nadia Hedhli, Chull Hong, Jing Liu, Guiping Yang, Li Wang, Huacheng Dai, Univ. Medicine New Jersey, Newark, NJ; Thomas Wagner, Greenville Hosp System, Greenville, SC; Stephen F Vatner; Univ. Medicine New Jersey, Newark, NJ H11 kinase is a protein of unknown function mainly expressed in heart and skeletal muscle. Its expression is markedly increased in the ischemic heart together with an array of cytoprotective and anti-apoptotic genes, which supposes that H11 kinase promotes cell survival. Paradoxically, overexpression of H11 kinase in other cell types induces apoptosis. Therefore, the goal of the present study was to determine how H11 kinase affects cardiac cell survival and death, using both a cardiac-specific transgenic (TG) mouse model and cardiac cell culture. After 45 min coronary occlusion in vivo, infarct size in hearts from TG mice (7-fold overexpression) was reduced by 80% compared to wild type (P<0.01). This protection was related to a significant (P<0.05) increase in the expression of proteins promoting cell survival, including heat-shock proteins (HSP70, mmDJA4), phospho-Akt, the mTOR targets p70S6K and 4EBP-1, and the glucose transporter GLUT1. Surprisingly, although overexpression of H11 kinase is protective in the TG animal, ischemia reduced H11 kinase expression by 60% in wild type mice (P<0.01 versus sham). To reconcile this paradox, we performed a subcellular fractionation, which revealed that H11 kinase is expressed in both the cytosol and the nucleus. In wild type mice, the cytosolic expression of H11 kinase during ischemia was reduced by more than 80%, whereas the nuclear expression was unaffected. In the TG mouse, overexpression of H11 kinase accumulated as a nuclear protein, which therefore mediates the survival effect. To understand the role of the cytosolic form of the protein, we transfected isolated cardiac myocytes with increasing doses of adenovirus (3 MOI to 100 MOI) harboring the …

Production of human clotting Factor IX without toxicity in mice after vascular delivery of a lentiviral vector

Replication-deficient lentiviral vectors (LV) have been shown to enable the stable genetic modification of multiple cell types in vivo. We demonstrate here that vascular and hepatic delivery of a third-generation HIV-derived lentiviral vector encoding human Factor IX (LV-hFIX) produced potentially therapeutic serum levels of hFIX protein with no vector-mediated local or systemic toxicity of adult mice. Portal vein administration produced the highest serum levels of hFIX and demonstrated proportionally higher levels of gene transfer to the liver with up to 4% of hepatocytes expressing hFIX. Vascular delivery of a lentiviral vector encoding GFP resulted in genetic modification of up to 12% of liver cells. Cell proliferation was not required for hepatocyte transduction with either vector. Serum hFIX levels reached 4% of normal levels following vascular LV-mediated hFIX gene transfer and remained stable for months following vector administration.