Optimizing lentiviral vector transduction of hematopoietic stem cells for gene therapy.

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34+ cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34+ cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34+ cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34+ cell concentrations (2-4 × 106/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34+ cell transduction with clinically relevant LVs.

Genetic alteration of SJ293TS cells and modification of serum-free media enhances lentiviral vector production.

Successful cell and gene therapy clinical trials have resulted in the US Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies, which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells, have driven additional investigations, increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods, we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media, SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold, while absence of Beta-2 microglobulin, a key component of major histocompatibility complex class I molecules, has been reported to reduce the immunogenicity of lentiviral particles. Furthermore, we describe an improved methodology for culturing SJ293TS-DPB that facilitates expansion, reduces handling, and increases titers by 2-fold compared with previous methods. SJ293TS-DPB stably produced lentiviral vectors for over 4 months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34+ hematopoietic stem cells.

Transduction of human primitive repopulating hematopoietic cells with lentiviral vectors pseudotyped with various envelope proteins.

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34(+) peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34(+) cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34(+) cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45(+) cells in total bone marrow were comparable to that of the control, mock-transduced group (37-45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the gamma-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the gamma-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.

Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media.

Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Cholesterol dependence of HTLV-I infection.

Cholesterol-rich plasma membrane microdomains are important for entry of many viruses, including retroviruses. Depletion of cholesterol with 2-hydroxypropyl-beta-cyclodextrin inhibits entry of human T cell leukemia virus type I (HTLV-1) and HTLV-I envelope pseudotyped lentivirus particles. Using a soluble fusion protein of the HTLV-I surface envelope protein with the immunoglobulin Fc domain, the HTLV-I receptor was found to colocalize with a raft-associated marker and to cluster in specific plasma membrane microdomains. Depletion of cholesterol did not alter receptor binding activity, suggesting a requirement for cholesterol in a postbinding virus entry step.

Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy.

We have developed a producer cell line that generates lentiviral vector particles of high titer. The vector encodes the Wiskott-Aldrich syndrome (WAS) protein. An insulator element has been added to the long terminal repeats of the integrated vector to limit proto-oncogene activation. The vector provides high-level, stable expression of WAS protein in transduced murine and human hematopoietic cells. We have also developed a monoclonal antibody specific for intracellular WAS protein. This antibody has been used to monitor expression in blood and bone marrow cells after transfer into lineage negative bone marrow cells from WAS mice and in a WAS negative human B-cell line. Persistent expression of the transgene has been observed in transduced murine cells 12-20 weeks following transplantation. The producer cell line and the specific monoclonal antibody will facilitate the development of a clinical protocol for gene transfer into WAS protein deficient stem cells.

Comparison of insulators and promoters for expression of the Wiskott-Aldrich syndrome protein using lentiviral vectors.

Gene therapy for the treatment of Wiskott-Aldrich syndrome (WAS) presents an alternative to the current use of allogeneic bone marrow transplantation. We describe the development of a self-inactivating lentiviral vector containing chromatin insulators for treatment of WAS and compare a gammaretroviral (MND), human cellular (EF1α), and the human WASp gene promoter for expression patterns in vivo during murine hematopoiesis using the green fluorescent protein (GFP) marker. Compared with the EF1α and the WASp promoters, expression from the MND promoter in mouse transplant recipients was much higher in all lineages examined. Importantly, there was sustained expression in the platelets of secondary recipient animals, necessary to correct the thrombocytopenia defect in WAS patients. Analysis of WAS protein expression in transduced human EBV-immortalized B-cells and transduced patient peripheral blood mononuclear cells also demonstrated stronger expression per copy from the MND promoter compared with the other promoters. In addition, when analyzed in an LM02 activation assay, the addition of an insulator to MND-promoter-containing constructs reduced transactivation of the LM02 gene. We propose a clinical trial design in which cytokine-mobilized, autologous, transduced CD34(+) cells are administered after myelosuppression.

Suppression of clonal dominance in cultured human lymphoid cells by addition of the cHS4 insulator to a lentiviral vector.

Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood dis-orders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hy-persensitive site 4 insulator from the chicken beta-globin locus (cHS4) possesses chromatin bar-rier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 insulator element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among indi-vidual single cell isolates. Most unique integration sites were within genes, but the insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal domi-nance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of mul-tiple single cell isolates containing the same integration site. We infer that certain integrations conferred a prolifera-tive or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the insulator element in the vector genome.