Simian Immunodeficiency Virus Vector Research Articles

Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages.

BackgroundThe cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases, which sulfonate proteins, glycoproteins, glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs), which sulfonate various small molecules including hormones, neurotransmitters, and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions, cell signaling, and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism, namely reverse transcription.MethodsMDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions, MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels, and qPCR was used to measure viral RNA and DNA products.ResultsWe demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs, and through siRNA knockdown experiments, we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription.ConclusionsThese studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules, raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0491-9) contains supplementary material, which is available to authorized users.

Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

Production of functional coagulation factor VIII from iPSCs using a lentiviral vector

The use of induced pluripotent stem cells (iPSCs) as an autologous cell source has shed new light on cell replacement therapy with respect to the treatment of numerous hereditary disorders. We focused on the use of iPSCs for cell-based therapy of haemophilia. We generated iPSCs from mesenchymal stem cells that had been isolated from C57BL/6 mice. The mouse iPSCs were generated through the induction of four transcription factor genes Oct3/4, Klf-4, Sox-2 and c-Myc. The derived iPSCs released functional coagulation factor VIII (FVIII) following transduction with a simian immunodeficiency virus vector. The subcutaneous transplantation of iPSCs expressing FVIII into nude mice resulted in teratoma formation, and significantly increased plasma levels of FVIII. The plasma concentration of FVIII was at levels appropriate for human therapy at 2-4 weeks post transplantation. Our data suggest that iPSCs could be an attractive and prospective autologous cell source for the production of coagulation factor, and that engineered iPSCs expressing coagulation factor might provide a cell-based therapeutic strategy appropriate for haemophilia.

High-efficiency Transduction of Rhesus Hematopoietic Repopulating Cells by a Modified HIV1-based Lentiviral Vector

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34(+) cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34(+) cells were transduced with standard SIV vectors and the other half with χHIV vectors. As compared with SIV vectors, χHIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of χHIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, χHIV vector frequently integrated into gene regions, especially into introns. In summary, our χHIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This χHIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models.

Intra‐articular injection of mesenchymal stem cells expressing coagulation factor ameliorates hemophilic arthropathy in factor VIII‐deficient mice

Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor-1 (PAI-1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI-1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI-1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII-deficient mice. Interestingly, intra-articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII-deficient mice. The therapeutic effects of a single intra-articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. MSCs provide a promising autologous cell source for the production of coagulation factor. Intra-articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.

BioKnife, a uPA Activity-dependent Oncolytic Sendai Virus, Eliminates Pleural Spread of Malignant Mesothelioma via Simultaneous Stimulation of uPA Expression

Malignant pleural mesothelioma (MPM) is highly intractable and readily spreads throughout the surface of the pleural cavity, and these cells have been shown to express urokinase-type plasminogen activator receptor (uPAR). We here examined the potential of our new and powerful recombinant Sendai virus (rSeV), which shows uPAR-specific cell-to-cell fusion activity (rSeV/dMFct14 (uPA2), named "BioKnife"), for tumor cell killing in two independent orthotopic xenograft models of human. Multicycle treatment using BioKnife resulted in the efficient rescue of these models, in association with tumor-specific fusion and apoptosis. Such an effect was also seen on both MSTO-211H and H226 cells in vitro; however, we confirmed that the latter expressed uPAR but not uPA. Of interest, infection with BioKnife strongly facilitated the uPA release from H226 cells, and this effect was completely abolished by use of either pyrrolidine dithiocarbamate (PDTC) or BioKnife expressing the C-terminus-deleted dominant negative inhibitor for retinoic acid-inducible gene-I (RIG-IC), indicating that BioKnife-dependent expression of uPA was mediated by the RIG-I/nuclear factor-κB (NF-κB) axis, detecting RNA viral genome replication. Therefore, these results suggest a proof of concept that the tumor cell-killing mechanism via BioKnife may have significant potential to treat patients with MPM that is characterized by frequent uPAR expression in a clinical setting.

TRIM5α Variation Affects Lentiviral Transduction Efficiency for Long-Term Hematopoietic Repopulating Cells in a Rhesus Stem Cell Transplantation Model

Abstract 2056The Tripartite Motif-containing protein 5 α (TRIM5α) is thought to play an important role in restricting retroviral infection among species in nature, and restricts HIV-1 based lentiviral transduction of rhesus hematopoietic repopulating cells. We previously developed a chimeric HIV-1 based lentiviral vectors (χHIV vectors) which includes a simian immunodeficiency virus (SIV) capsid in place of the HIV-1 capsid to circumvent this restriction (J Virol. 2009). Transduction efficiency, however, remained highly variable between animals. In this study, we sought to evaluate the effects of rhesus TRIM5α polymorphisms on lentiviral transduction of rhesus hematopoietic repopulating cells.To evaluate whether rhesus TRIM5α polymorphisms influence the efficiency of lentiviral transduction, we transduced cell lines expressing 6 different rhesus TRIM5α types (Mamu-1, -2, -3, -4, -5, and TrimCyp) with eGFP-encoding HIV1, χHIV, and SIV vectors (Figure). Among all TRIM5α cell lines, transgene expression rates (%GFP) from the χHIV vector fell between that of the HIV-1 vector and that of the SIV vector. For the χHIV and SIV vectors, transduction efficiency was reduced in Mamu-1, -2, and -3 (p<0.01) expressing cell lines when compared to that of control cells. For the HIV-1 vector, there was a reduction in %GFP among all TRIM5α types (p<0.01). These results suggest that both the χHIV and SIV vectors escape restriction through rhesus TRIM5α Mamu-4, -5, and TrimCyp.We then analyzed 16 rhesus macaques who were transplanted with CD34+ cells transduced with the χHIV vector. We evaluated %GFP in granulocytes and lymphocytes 6 months after transplantation, as %GFP expression has been found to plateau in the peripheral blood at 6 months. Transduction efficiency of rhesus CD34+ cells was also evaluated in vitro at the time of transplant based on %GFP expression. For statistical analysis, we assessed factors that potentially affect transduction efficiency, including age, sex, weight, total number of mobilized CD34+ cells, cytokine mobilization regimen (G-CSF & stem cell factor (SCF) vs. G-CSF & plerixafor), cell density during transduction, and TRIM5α polymorphisms.Multivariable analysis demonstrated that TRIM5α type Mamu-4 (50.9±19.0% vs. 26.6±16.7%, p=0.04) as well as mobilization regimen (48.5±17.4% vs. 12.7±7.1%, p=0.01) affected CD34+ cell transduction efficiency in vitro. TRIM5α Mamu-4 only showed significant effects on %GFP among lymphocytes in vivo (23.7±17.9% vs. 5.3±3.1%, p=0.046). When analyzing the %GFP among granulocytes in vivo, there was a significant correlation with weight (p=0.01), mobilized CD34+ cell number (p=0.02), TRIM5α type Mamu-4 (29.3±25.4% vs. 8.4±6.0%, p=0.03), and mobilization regimen (25.4±24.1% vs. 7.5±3.0%, p=0.04). If in vitro %GFP is included in the analysis, both %GFP among granulocytes and lymphocytes are strongly affected (<0.001) and TRIM5α type Mamu-4 and mobilization regimen are no longer significant. Univariate analysis showed similar tendencies regarding %GFP among granulocytes and lymphocytes. Taken together, our data suggest that TRIM5α type Mamu-4, mobilization regimen (G-CSF & SCF), and CD34+ cell transduction efficiency in vitro are important factors that can predict higher %GFP in granulocytes and lymphocytes 6 months following transplantation.In summary, rhesus TRIM5α polymorphisms (especially type Mamu-4) play an important role in allowing efficient lentiviral transduction of long-term repopulating cells and contribute to the variable results observed in the rhesus hematopoietic stem cell gene therapy model. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High Transgene Expression Rates After Extended Follow up Among Rhesus Macaque Recipients of Autologous Hematopoietic Stem Cells Transduced with a Modified HIV1-Based Lentiviral Vector

Abstract 3118Hematopoietic stem cell (HSC)-targeted gene therapy is potentially curative for the hemoglobin disorders; however, highly efficient, lineage specific globin expression remains elusive, and large animal models thus remain important for further development toward clinical application.We previously constructed a chimeric HIV1 vector (χHIV vector) system to circumvent a species specific restriction to HIV1-based vectors wherein the HIV1 vector genome is packaged in the context of the simian immunodeficiency virus (SIV) capsid for efficient transduction of rhesus CD34+ cells in vitro (J Virol. 2009) and in vivo (ASH 2009). In this study, we sought to evaluate transduction efficiency and vector integration pattern among long-term repopulating cells in the rhesus HSC transplantation model.We followed up transgene expression rates among peripheral blood cells of three animals for 1.5–2 years. For two animals (RQ7307 and RQ7280), half of the CD34+ cells were transduced with a standard SIV vector and the other half with the χHIV vector using the same protocol. Transduced cells were transplanted into lethally irradiated rhesus macaques, as previously described (J Virol. 2009). The transgene expression rates in peripheral blood cells plateaued 3–4 months after transplantation and similar transgene expression rates continued in all cell lineages for at least 1.5 years (Figure). The χHIV vector demonstrated that 2–3 fold higher transgene expression rates were seen in granulocytes (RQ7307: 8.6±0.2% vs. 3.1±0.1%, RQ7280: 27.9±0.7% vs. 18.4±0.2%) and RBCs (RQ7307: 3.3±0.1% vs. 0.9±0.0%, RQ7280: 10.0±0.1% vs. 4.0±0.1%), and equivalent transgene expression rates in lymphocytes (RQ7307: 7.8±0.2% vs. 4.5±0.1%, RQ7280: 22.4±0.5% vs. 17.6±0.3%) and platelets (RQ7307: 3.1±0.1% vs. 2.7±0.1%, RQ7280: 12.3±0.2% vs. 16.8±0.2%), compared to the SIV vector. The average vector copy numbers in transduced cells were 4.6–5.7 for the χHIV vector and 1.5–2.0 for the SIV vector in both transplanted animals, evaluated by Southern blot analysis.We then performed transplantation of rhesus CD34+ cells which were transduced with the χHIV vector alone to evaluate transgene expression and vector integration pattern. Transgene expression rates among peripheral blood cells in this animal (RQ7387) plateaued 1–3 months after transplantation, with stable high transgene expression rates of 51.7±1.2% in granulocytes, 54.7±0.1% in lymphocytes, 22.1±0.2% in RBCs, and 19.1±0.1% platelets for 2 years after transplantation. Multi-lineage marking was observed by flow cytometric analysis.We then evaluated integration sites for the χHIV vector in the recipient of χHIV vector alone transduced cells by linear amplification mediated-PCR, using peripheral blood cells of RQ7387 in 0.5–1.5 years after transplantation. We found a total of 344 integration sites for the χHIV vector, and our data demonstrated that the χHIV vector integrated into gene regions, especially introns, when compared to the integration pattern of computer-generated random controls (p<0.001). On the other hand, our data revealed fewer integrations of the χHIV vector into ≤30kb upstream of genes (p<0.001) and into the upstream regions of transcription start sites. Most of the integration sites had low gene density (0–10 genes within 1 Mb upstream or downstream of integration sites, p<0.01), compared to that of random controls. No specific trend was noted for the number of integration sites around CpG islands and the number of CISs around integration sites. These data suggest that the χHIV vector has integration patterns comparable to HIV1 and SIV vectors.In summary, our χHIV vector shows efficient transduction for rhesus long-term repopulating cells, achieving sufficient levels for therapeutic effects in gene therapy trials for globin disorders. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Pigment Epithelium-Derived Factor Gene Therapy Targeting Retinal Ganglion Cell Injuries: Neuroprotection against Loss of Function in Two Animal Models

Lentiviral vectors are promising tools for the treatment of chronic retinal diseases including glaucoma, as they enable stable transgene expression. We examined whether simian immunodeficiency virus (SIV)-based lentiviral vector-mediated retinal gene transfer of human pigment epithelium-derived factor (hPEDF) can rescue rat retinal ganglion cell injury. Gene transfer was achieved through subretinal injection of an SIV vector expressing human PEDF (SIV-hPEDF) into the eyes of 4-week-old Wistar rats. Two weeks after gene transfer, retinal ganglion cells were damaged by transient ocular hypertension stress (110 mmHg, 60 min) and N-methyl-d-aspartic acid (NMDA) intravitreal injection. One week after damage, retrograde labeling with 4',6-diamidino-2-phenylindole (DAPI) was done to count the retinal ganglion cells that survived, and eyes were enucleated and processed for morphometric analysis. Electroretinographic (ERG) assessment was also done. The density of DAPI-positive retinal ganglion cells in retinal flat-mounts was significantly higher in SIV-hPEDF-treated rats compared with control groups, in both transient ocular hypertension and NMDA-induced models. Pattern ERG examination demonstrated higher amplitude in SIV-hPEDF-treated rats, indicating the functional rescue of retinal ganglion cells. These findings show that neuroprotective gene therapy using hPEDF can protect against retinal ganglion cell death, and support the potential feasibility of neuroprotective therapy for intractable glaucoma.

Production of SIV Vectors for Gene Delivery

INTRODUCTIONThe acquisition of vectors suitable for in vivo gene delivery has been a recurrent theme in gene therapy research. Several challenging hurdles must be overcome to reach this goal. First, the vectors must be prepared at high titers. Second, to avoid inactivation, the gene-transfer vectors should not be recognized by the host immune system. Third, the vectors should be able to replicate and to express a transgene in cells that are nonproliferating or slowly proliferating, a predominant situation in vivo. Lentiviral vectors derived from simian immunodeficiency virus (SIV) are similar to those derived from human immunodeficiency virus (HIV-1 or HIV-2) in that they can insert transgenes in nonproliferating cells. It is becoming clear, however, that SIV vectors perform better than HIV-1 vectors in simian cells, and thus may be a valid alternative to HIV-1-based vectors in the early phases of the clinical testing of lentiviral vectors in nonhuman primate models. This protocol describes the production of SIV vector particles and the assay used to measure transduction efficiency. Included also are methods for detecting replication-competent retroviruses (RCRs) and testing the putative mobilization of the green fluorescent protein (GFP)-containing SIV vector. The percentage of GFP-positive cells is determined by fluorescence-activated cell sorting (FACS). These safety assays make use of sMAGI target cells that are permissive to SIVmac251 replication and harbor a stably integrated lacZ transcription unit driven by the SIV/HIV tat-inducible HIV-1 long terminal repeat (LTR).

Development of a lentiviral vector system to study the role of the Andes virus glycoproteins

To infect target cells, enveloped viruses use their virion surface proteins to direct cell attachment and subsequent entry via virus-cell membrane fusion. How hantaviruses enter cells has been largely unexplored. To study early steps of Andes virus (ANDV) cell infection, a lentiviral vector system was developed based on a Simian immunodeficiency virus (SIV) vector pseudotyped with the ANDV-Gn/Gc envelope glycoproteins. The incorporation of Gn and Gc onto SIV-derived vector particles was assessed using newly generated monoclonal antibodies against ANDV glycoproteins. In addition, sera of ANDV infected humans were able to block cell entry of the SIV vector pseudotyped with ANDV glycoproteins, suggesting that their antigenic conformation is similar to that in the native virus. The use of such SIV vector pseudotyped with ANDV-Gn/Gc glycoproteins should facilitate studies on ANDV cell entry. Along this line, it was found that depletion of cholesterol from target cells strongly diminished cell infection, indicating a possible role of lipid rafts in ANDV cell entry. The Gn/Gc pseudotyped SIV vector has several advantages, notably high titer vector production and easy quantification of cell infection by monitoring GFP reporter gene expression by flow cytometry. Such pseudotyped SIV vectors can be used to identify functional domains in the Gn/Gc glycoproteins and to screen for potential hantavirus cell entry inhibitors.

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.

A Modified HIV1-Based Lentiviral Vector Can Transduce Rhesus Hematopoietic Repopulating Cells as Efficiently as An SIV Vector in An Autologous Transplantation Model.

Abstract 692HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press).To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p<0.01, second rhesus: 65.0±0.51% vs. 77.0±0.18%, p<0.01, respectively). Following transplantation and reconstitution, however, the χHIV vector showed modestly higher gene marking levels in granulocytes (first rhesus: 12.4% vs. 6.1%, second rhesus: 36.1% vs. 27.2%) and equivalent marking levels in lymphocytes, red blood cells (RBC), and platelets, compared to the SIV vector at one month (Figure). Three to four months after transplantation in the first animal, in vivo marking levels plateaued, and the χHIV achieved 2-3 fold higher marking levels when compared to the SIV vector, in granulocytes (6.9% vs. 2.8%) and RBCs (3.3% vs. 0.9%), and equivalent marking levels in lymphocytes (7.1% vs. 5.1%) and platelets (2.8% vs. 2.5)(Figure). Using cell type specific surface marker analysis, the χHIV vector showed 2-7 fold higher marking levels in CD33+ cells (granulocytes: 5.4% vs. 2.7%), CD56+ cells (NK cells: 6.5% vs. 3.2%), CD71+ cells (reticulocyte: 4.5% vs. 0.6%), and RBC+ cells (3.6% vs. 0.9%), and equivalent marking levels in CD3+ cells (T cells: 4.4% vs. 3.3%), CD4+ cells (T cells: 3.9% vs. 4.6%), CD8+ cells (T cells: 4.2% vs. 3.9%), CD20+ cells (B cells: 7.6% vs. 4.8%), and CD41a+ cells (platelets: 3.5% vs. 2.2%) 4 months after transplantation. The second animal showed a similar pattern with higher overall levels (granulocytes: 32.8% vs. 19.1%, lymphocytes: 24.4% vs. 17.6%, RBCs 13.1% vs. 6.8%, and platelets: 14.8% vs. 16.9%) 2 months after transplantation.These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High-Expression Porcine FVIII Driven by Erythroid-Specific Promoters for Lentiviral Vector-Mediated Gene Therapy

Hemophilia A is an X-linked gene disorder that results in a deficiency of circulating coagulation factor VIII (fVIII) and may be ameliorated by only modest amounts of circulating protein, which makes it a logical candidate for gene therapy. Due to the potential risk of insertional mutagenesis from oncoretroviral-mediated gene therapy, cell-specific expression of transgenes using self-inactivating viral vectors may provide a safer gene therapy approach for use in humans. Therefore, we constructed simian immunodeficiency virus (SIV)-based lentiviral vectors containing a 5′ long-terminal repeat (LTR) and 3′ LTR with self-inactivating U3 deletion, the bovine growth hormone polyA signal, a packaging signal (ψ), and a single internal ankyrin-1 or β-globin promoter, designated SIV-Ank and SIV-Bg, respectively. The minimal 314-bp ankyrin-1 promoter and 180-bp β-globin promoter flanked upstream by enhancing sequences, HS2, HS3, and HS4 (Hanawa et al., Hum Gene Ther, 2002) from the locus control region were cloned into the SIV vector backbone upstream from either enhanced green fluorescent protein (eGFP) or B-domain deleted porcine factor VIII (BDDpfVIII). The erythroid-specificity of each promoter was evaluated in vitro by measurement of either eGFP or fVIII expression following transduction of SIV-Ank and SIV-Bg constructs into both K562 myelogenous leukemic cells and 293T human embryonic kidney cells. GFP expression, as measured by flow cytometry, in transduced cells revealed that the ankyrin-1 and β-globin promoters are more active in K562 cells as compared to 293T cells. The β-globin promoter yielded higher mean fluorescent intensity values for GFP compared to the ankyrin-1 promoter at similar MOIs in K562 cells, suggesting stronger β-globin promoter activity in these cells. Transduction of cells with the SIV vector encoding BDDpfVIII driven by the β-globin promoter resulted in a 14-fold higher number of transcripts per DNA copy number in K562 cells compared to 293T cells, while cells transduced with the ankyrin-l promoter had only a 1.4-fold greater number of transcripts per DNA copy number. In addition, SIV-Bg-fVIII-modified K562 cells produced a 5.2-fold greater number of transcripts per DNA copy number than SIV-Ank- fVIII-modified cells. To evaluate the usefulness of these vectors for in vivo expression of BDDpfVIII, hemophilia A mice (exon 16 knockout) were conditioned with 11 Gy total body irradiation and transplanted with gene-modified Sca-1+ cells transduced with either SIV-Ank-fVIII, SIV-Ank-eGFP, SIV-Bg-fVIII, or SIV-Bg-eGFP. The expression of eGFP from donor red blood cells in recipient mice was approximately 8–12% using both the ankyrin-1 and β-globin promoter constructs. Mice that received cells transduced with SIVAnk- fVIII demonstrated therapeutic levels of plasma fVIII up to 0.5 units/mL (i.e. 50% normal human levels). However, fVIII expression decreased over time and real-time PCR analysis of peripheral blood cells confirmed the loss of detectable fVIII transgene by 6 weeks after transplantation, suggesting there was predominantly gene transfer into short-term repopulating hematopoietic cells. Mice transplanted with SIV-Bg-fVIII-modified hematopoietic stem cells demonstrated a similar rise and fall of fVIII expression within the first 4 weeks after transplantation, and showed an increase in fVIII expression by 6 weeks. At 8 weeks post transplantation, fVIII levels greater than 300% normal human levels were observed. Red blood cell count, hemoglobin, and red blood cell morphology were normal despite the high level of expression of fVIII. Overall these data demonstrate the potential for therapeutic expression of factor VIII using a self-inactivating lentiviral vector containing an erythroid-specific internal promoter.

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Phenotypic Correction of Hemophilia A by Ectopic Expression of Activated Factor VII in Platelets

Platelets are receiving much attention as novel target cells to secrete a coagulation factor for hemophilia gene therapy. In order to extend the application of platelet-directed gene therapy, we examined whether ectopic expression of activated factor VII (FVIIa) in platelets would result in an efficient bypass therapy to induce sufficient thrombin generation on platelet surfaces in mice with hemophilia A. Transduction of bone marrow cells with a simian immunodeficiency virus (SIV)-based lentiviral vector harboring the platelet-specific GPIb alpha promoter resulted in efficient transgene expression in platelets. FVIIa antigen was expressed in platelets by this SIV system; FVII transgene products were found to localize in the cytoplasm and translocate toward the sub-membrane zone and cell surface after activation. Although FVII antigen levels in platelets did not reach the therapeutic levels seen with FVIIa infusion therapy, whole-blood coagulation, as assessed by thromboelastography, was significantly improved in mice with hemophilia A. Further, we observed correction of the bleeding phenotype in mice with hemophilia A after transplantation, even in the presence of FVIII-neutralizing antibodies. Our results demonstrate that FVIIa-expressing platelets can strengthen hemostatic function and may be useful in treating hemophilia and other inherited bleeding disorders. These findings are comparable to the proven therapeutic effects of FVIIa infusion.

Simian immunodeficiency virus vector pseudotypes differ in transduction efficiency and target cell specificity in brain

Lentiviral vectors have proven to be promising tools for transduction of brain cells in vivo and in vitro. In this study, we have examined the central nervous system (CNS) transduction efficiencies and patterns of a self-inactivating simian immunodeficiency virus (SIVmac)-derived lentiviral vector pseudotyped with glycoproteins from the vesicular stomatitis virus (VSV-G), the amphotropic murine leukemia virus (MLV4070Aenv), the lymphocytic choriomeningitis virus (LCMV-GP), the Ross River virus (RRV-GP) and the rabies virus (RV-G). All glycoproteins were efficiently incorporated into SIV virions, allowing efficient transduction of neuronal cell lines as well as of primary dissociated mouse brain cell cultures. After injection of highly concentrated vector stocks into the striatum of adult mice, quantitative analyses revealed high transduction efficiency with VSV-G pseudotypes, while LCMV-GP and RV-G pseudotypes exhibited moderate transduction efficiencies. MLV4070Aenv and RRV-GP pseudotypes, however, showed only weak levels of transduction after stereotactic injection into the brain. Regarding cell tropism in vivo, VSV-G-pseudotyped SIV vectors transduced neuronal as well as glial cells, whereas all other pseudotypes preferentially transduced neuroglial cells. In addition, we analyzed the influence of the central polypurine tract (cPPT) in context of the VSV-G-pseudotyped SIV transfer vector for infection of brain cells. Deletion of the cPPT sequence from the transfer vector decreased the in vivo transduction efficiency by fourfold, and, more importantly, this modification changed the transduction pattern, since these vectors were no longer able to infect neuronal cells in vivo. Vector injection into the brain did elicit a humoral immune response in the injected hemisphere; however, no gross signs of inflammation could be detected. Analysis of the biodistribution of the vector revealed that, besides the injected brain region, no vector-specific sequences could be detected in any of the organs evaluated. These data indicate SIV vectors as efficient gene delivery vehicles for the treatment of neurodegenerative diseases.

Potent Inhibition of Simian Immunodeficiency Virus (SIV) Replication by an SIV-Based Lentiviral Vector Expressing Antisense Env

In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.

An Antiproliferative Genetic Screening Identifies a Peptide Aptamer That Targets Calcineurin and Up-regulates Its Activity

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.

Retroviral Vector Integration Site Analysis in Rhesus Macaques Transplanted with Hematopoietic Stem Cells Transduced with Simian Immunodeficiency Virus Vectors.

Lentiviral vectors derived from the Simian Immunodeficiency Virus (SIV) mediate relatively efficient transduction of hematopoietic stem cells (HSCs) from rhesus macaques. While integration sites associated with onco-retroviral vectors have been extensively studied in primate transplantation experiments, much less in known about lentiviral vector integration site patterns. The existing literature is limited to one report showing that SIV vectors have a distinctive genomic integration pattern compared with onco-retroviral vectors (Hematti et al 2004). Here we report our results mapping 263 integration sites for SIV vectors in an autologous rhesus macaque transplantation model. Two SIV vectors were used that expressed either MGMT-P140K alone or MGMT-P140K together with HOXB4 from an internal MSCV promoter. Two rhesus macaques were transplanted with autologous CD34+ cells, half of which were transduced with the MGMT vector and half were transduced with MGMT-HOXB4 vector. The first animal was treated with 7 courses of temozolomide and 6-BG which has resulted in selection of transduced cells in vivo, both at the level of myeloid progenitors, and to a lesser degree, in HSCs. A total of 152 integration sites were identified from this animal based on LAM-PCR. Sequence analysis showed a favored preference for integration into transcription units, which comprised 70% of all integrations, with 64% integrations occurring within introns and 6% within exons. The highest density of SIV integration sites per Mbp were on chromosomes 17 and 19 (0.17 and 0.2 respectively). At different time points during drug treatment, multiple clones contributed to hematopoiesis and 24 clones were identified repetitively. The second animal was treated with two courses of TMZ/BG and two courses of BCNU/BG resulting in selection of transduced cells in all lineages. So far, a total of 111 integration sites have been identified in this animal and a similar general integration pattern was observed as seen in the first animal. Integration into transcription units was favored (71%) with 65% occurring within introns and 6% within exons. The three most gene-dense chromosomes 17, 19 and 22 had the highest density of SIV integration sites (0.11, 0.16 and 0.18 respectively). In this animal, 10 out 111 integration sites were identified repetitively during the drug treatments. Vector integrations near previously described oncogenes were identified in both animals (19 out 152 and 11 out of 111 integration sites for each animal respectively). However, no common integration sites (CIS) into a single oncogene were observed and no abnormal hematopoietic proliferation developed in either animal. Moreover, there were no integrations seen within the MDS/Evi locus that has been previously shown to be a CIS for onco-retroviral vectors. Our study shows that the SIV integration pattern is distinctly different from that obtained with murine oncoretroviral vectors and is consistent with the previous study. The lack of integrations within the MDS1/Evi locus represents a potential safety advantage, however further study will be necessary to determine whether the overall propensity for insertional mutagenesis and transformation is decreased. We also show that multiple clones contributed to hematopoiesis before and after MGMT-mediated selection suggesting that this approach is not necessarily associated with restrictions in clonal numbers contributing to hematopoiesis.