Although pancreatic beta-cell transplantation may serve as a potential cure for diabetes mellitus (DM), limited donor tissue availability poses a major challenge. Thus, there is a great demand to find new sources for pancreatic beta-cells. Here, we present a lentiviral vector-based approach to achieve beta-cell proliferation through the beta-cell-specific activation of the hepatocyte growth factor (HGF)/cmet signaling pathway. The methodology is based on the beta-cell-specific expression of a ligand-inducible, chimeric receptor (F36Vcmet), under transcriptional control of the promoter from the human insulin gene, and its ability to induce HGF/cmet signaling in the presence of a synthetic ligand (AP20187). High transduction efficiency of human pancreatic islets was achieved utilizing this approach with chimeric receptor expression confined to the beta-cell population. In addition, specific proliferation of human pancreatic beta-cells was induced utilizing this approach. Selective, regulated beta-cell expansion may help to provide greater availability of cells for transplantation in patients with DM.

We investigated the use of integrase-defective lentiviral vectors (IDLVs) for transient delivery of zinc finger nucleases (ZFNs) and donor templates for site-specific modification of the human adenosine deaminase (hADA) gene. Initially, we constructed IDLVs carrying ZFN monomers (Single-IDLVs) and found them to be able to deliver their gene-editing payload to K562 cells successfully upon cotransduction, with minimal cytotoxicity. To simplify delivery, we designed an IDLV construct to deliver both ZFN monomers from the same vector (Double-IDLV). However, this construct in its original state was prone to rearrangements of the vector genome, resulting in greatly reduced functionality; this was due to recombination between highly similar ZFN monomers arranged in tandem. We modified the Double-IDLV constructs to reduce recombination and restored simultaneous delivery of both ZFNs. We also tested an IDLV construct for delivery of donor templates and demonstrated its efficacy for gene modification. In summary, we highlighted the importance of modifying vector design for co-delivery of highly similar sequences inherent to genome-editing nucleases, and demonstrated significant improvement in the use of IDLVs for delivery of ZFNs and donor templates for genome modification.

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 108 to 1 × 109 CD34+ cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34+ cells uses LV inefficiently, because the majority of CD34+ cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34+CD38- cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34+CD38- cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34+ purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34+CD38- cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38+ cells. Importantly, LV modification and transplantation of IB-purified CD34+CD38- cells/non-modified CD38+ cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34+ cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders (LSDs). The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin. Cystine accumulates in all tissues and leads to organ damage including end-stage renal disease. Using the Ctns(-/-) murine model for cystinosis, we tested the use of hematopoietic stem and progenitor cells (HSPC) genetically modified to express a functional CTNS transgene using a self-inactivating-lentiviral vector (SIN-LV). We showed that transduced cells were capable of decreasing cystine content in all tissues and improved kidney function. Transduced HSPC retained their differentiative capabilities, populating all tissue compartments examined and allowing long-term expression of the transgene. Direct correlation between the levels of lentiviral DNA present in the peripheral blood and the levels present in tissues were demonstrated, which could be useful to follow future patients. Using a new model of cystinosis, the DsRed Ctns(-/-) mice, and a LV driving the expression of the fusion protein cystinosin-enhanced green fluorescent protein (eGFP), we showed that cystinosin was transferred from CTNS-expressing cells to Ctns-deficient adjacent cells in vitro and in vivo. This transfer led to cystine decreases in Ctns-deficient cells in vitro. These data suggest that the mechanism of cross-correction is possible in cystinosis.

Serious adverse events in some human gene therapy clinical trials have raised safety concerns when retroviral or lentiviral vectors are used for gene transfer. We evaluated the potential for generating replication-competent retrovirus (RCR) and assessed the risk of occurrence of adverse events in an in vivo system. Human hematopoietic stem and progenitor cells (HSCs) and mesenchymal stem cells (MSCs) transduced with two different Moloney murine leukemia virus (MoMuLV)-based vectors were cotransplanted into a total of 481 immune-deficient mice (that are unable to reject cells that become transformed), and the animals were monitored for 18 months. Animals with any signs of illness were immediately killed, autopsied, and subjected to a range of biosafety studies. There was no detectable evidence of insertional mutagenesis leading to human leukemias or solid tumors in the 18 months during which the animals were studied. In 117 serum samples analyzed by vector rescue assay there was no detectable RCR. An additional 149 mice received HSCs transduced with lentiviral vectors, and were followed for 2-6 months. No vector-associated adverse events were observed, and none of the mice had detectable human immunodeficiency virus (HIV) p24 antigen in their sera. Our in vivo system, therefore, helps to provide an assessment of the risks involved when retroviral or lentiviral vectors are considered for use in clinical gene therapy applications.

The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.

Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA(-/-) mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(-/-) mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34(+) cells transduced with 1-5 × 10(7) TU/ml had 1-3 vector copies/cell and expressed 1-2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.

Using a mouse model of adenosine deaminase-deficient severe combined immune deficiency syndrome (ADA-deficient SCID), we have developed a noninvasive method of gene transfer for the sustained systemic expression of human ADA as enzyme replacement therapy. The method of delivery is a human immunodeficiency virus 1-based lentiviral vector given systemically by intravenous injection on day 1 to 2 of life. In this article we characterize the biodistribution of the integrated vector, the expression levels of ADA enzyme activity in various tissues, as well as the efficacy of systemic ADA expression to correct the ADA-deficient phenotype in this mouse model. The long-term expression of enzymatically active ADA achieved by this method, primarily from transduction of liver and lung, restored immunologic function and significantly extended survival. These studies illustrate the potential for sustained in vivo production of enzymatically active ADA, as an alternative to therapy by frequent injection of exogenous ADA protein.

β-globin lentiviral vectors (β-LV) have faced challenges in clinical translation for gene therapy of sickle cell disease (SCD) due to low titer and sub-optimal gene transfer to hematopoietic stem and progenitor cells (HSPCs). To overcome the challenge of preserving efficacious expression while increasing vector performance, we used published genomic and epigenomic data available through ENCODE to redefine enhancer element boundaries of the β-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a β-LV of reduced proviral length, termed CoreGA-AS3-FB, produced at higher titers and possessing superior gene transfer to HSPCs when compared to the full-length parental β-LV at equal MOI. At low vector copy number, vectors containing the ENCODE core sequences were capable of reversing the sickle phenotype in a mouse model of SCD. These studies provide a β-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply.

Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.

Immune responses to transgene products may lead to rejection of transduced cells, limiting successful gene therapy for genetic diseases. While moderate dosages of chemotherapeutic agents such as busulfan may increase hematopoietic stem cells (HSC) engraftment, they are not immune suppressive and do not abrogate immune responses to transgene products. Studies focused on nonmyeloablative conditioning with busulfan ± fludarabine in a clinically relevant monkey model to induce immune suppression to allow cells expressing a foreign transgene product to persist. Bone marrow CD34(+) HSC were transduced in two equal fractions using simian immunodeficiency virus (SIV)-based lentiviral vectors carrying a nonexpressed DNA sequence tag (NoN) and the green fluorescent protein (GFP) reporter gene. Post-transplant there was no evidence of elimination of cells containing the potentially immunogenic GFP gene; several recipients had stable persistence of cells, and no differences were detected with fludarabine, which was rapidly cleared. Antibodies and cellular immune responses to GFP developed in recipients with the highest levels of GFP-marked cells, although these cells were not eliminated. These studies establish a clinically relevant pediatric primate model to assess the effects of conditioning regimens on the engraftment of transduced HSC and the immune responses to cells expressing a foreign gene product.