Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

Improved Titer and Gene Transfer by Lentiviral Vectors Using Novel, Small ß-Globin Locus Control Region Elements.

ß-globin lentiviral vectors (ß-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 ß-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a ß-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 ß-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 ß-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply.

Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the ß-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the ß-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.

Preclinical demonstration of lentiviral vector-mediated correction of immunological and metabolic abnormalities in models of adenosine deaminase deficiency.

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.

Integrase-defective lentiviral vectors as a delivery platform for targeted modification of adenosine deaminase locus.

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.

Allelic exclusion and peripheral reconstitution by TCR transgenic T cells arising from transduced human hematopoietic stem/progenitor cells.

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34(+) HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8(+) and CD4(+) T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4-5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8(+) and CD4(+) T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34(+) HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-ß genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.

Regulated expansion of human pancreatic beta-cells.

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.

Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells.

Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success.

Creating New ß-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human ß-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic ßAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.

Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice.

UNLABELLED: Methionine adenosyltransferase (MAT) is an essential enzyme that catalyzes the biosynthesis of S-adenosylmethionine. Hepatic MAT activity falls in chronic liver diseases, and mice lacking Mat1a are predisposed to liver injury and develop hepatocellular carcinoma (HCC) spontaneously by 18 months. The current work examined the hypothesis that liver cancer stem cells contribute to HCC in this model. Livers from 6- and 18-month-old Mat1a-knockout (KO) mice and their wild-type (WT) littermates were fractionated and isolated by flow cytometry. CD45- nonparenchymal (NP) cells were cultured using liver stem cell conditions. Cells were analyzed by real-time PCR and fluorescent immunohistochemistry (FIHC). Tumor formation was assessed by injecting 1 x 10(6) CD133+CD49f+ cells intraperitoneally into immune-deficient mice. The proportion of CD49f+ and CD133+ cells in the CD45-NP fraction increased 4.5- to 5.5-fold from 6 to 18 months in KO mice but not in their WT littermates. Compared to CD49f- cells from old KO mice, CD49f+ cells from the same animals had a markedly increased expression of several oncogenes. CD133+ cells with CD49f coexpression were selected in vitro and exhibited rapid growth, with the expression of biliary cytokeratins, alpha-fetoprotein, and c-Met by FIHC. Clonal expansion of single CD133+CD49f+ cells revealed maintenance of bipotency. After CD133+CD49f+ cells were injected into immune-deficient mice, 3 of the 8 mice developed tumors of liver epithelial cells after 6-8 weeks. CONCLUSION: Mat1a(-/-) mice have expansion of liver stem cells as they age. These cells have increased expression of several oncogenes and are tumorigenic in vivo. This is the first demonstration of adult liver stem cells possessing tumorigenic potential without the use of a carcinogen or manipulation of tumor-suppressor or oncogene expression.

A CD133-expressing murine liver oval cell population with bilineage potential.

Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

The human ankyrin 1 promoter insulator sustains gene expression in a ß-globin lentiviral vector in hematopoietic stem cells.

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the ß-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human ß-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term ß-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

ß-globin gene transfer to human bone marrow for sickle cell disease.

Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-ßAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-ßAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total ß-globin-like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2-3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-ßAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling ß-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells.

Highly efficient large-scale lentiviral vector concentration by tandem tangential flow filtration.

Large-scale lentiviral vector (LV) concentration can be inefficient and time consuming, often involving multiple rounds of filtration and centrifugation. This report describes a simpler method using two tangential flow filtration (TFF) steps to concentrate liter-scale volumes of LV supernatant, achieving in excess of 2000-fold concentration in less than 3h with very high recovery (>97%). Large volumes of LV supernatant can be produced easily through the use of multi-layer flasks, each having 1720 cm(2) surface area and producing ∼560 mL of supernatant per flask. Combining the use of such flasks and TFF greatly simplifies large-scale production of LV. As a demonstration, the method is used to produce a very high titer LV (>10(10)TU/mL) and transduce primary human CD34+ hematopoietic stem/progenitor cells at high final vector concentrations with no overt toxicity. A complex LV (STEMCCA) for induced pluripotent stem cell (iPSC) generation is also concentrated from low initial titer and used to transduce and reprogram primary human fibroblasts with no overt toxicity. Additionally, a generalized and simple multiplexed real-time PCR assay is described for lentiviral vector titer and copy number determination.

The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3.

The common cytokine receptor gamma chain (gamma c), an essential component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. Recently, a novel lymphokine (IL-21) and its receptor (IL-21R alpha) were described which profoundly affect the growth and activation state of B, T, and NK cells in concert with other lymphokines or stimuli [Parrish-Novak, J., et al. (2000) Nature 408, 57-63]. In this report, we show that gamma c is also a required signaling component of the IL-21 receptor (IL-21R) using the gamma c-deficient X-linked severe combined immunodeficiency (XSCID) lymphoblastoid cell line JT, and JT cells reconstituted with gamma c (JT/gamma c). Moreover, we demonstrate a functional requirement for both gamma c and the gamma c-associated Janus family tyrosine kinase 3 (JAK3) in IL-21-induced proliferation of pro-B-lymphoid cells engineered to express human IL-21R alpha (BaF3/IL-21R alpha). Retroviral-mediated transduction of wild-type gamma c into XSCID JT cells restored function to the IL-21R, as shown by IL-21-induced tyrosine phosphorylation of JAK1 and JAK3, and downstream activation of STAT5, in JT/gamma c cells as well as BaF3/IL-21R alpha and primary splenic B cells. In contrast, IL-21 failed to activate the JAK-STAT pathway in nonreconstituted JT cells. Monoclonal antibodies specific for the gamma c chain effectively inhibited IL-21-induced growth of BaF3/IL-21R alpha cells, supporting a functional role for this molecule in the IL-21R complex. In addition, the specific JAK3 tyrosine kinase inhibitor WHI-P131 significantly reduced IL-21-induced proliferation of BaF3/IL-21R alpha cells. Taken together, these results definitively demonstrate that IL-21-mediated signaling requires the gamma c chain, and indicate that JAK3 is an essential transducer of gamma c-dependent survival and/or mitogenic signals induced by this cytokine.