Safety and Efficacy of a Lentiviral Vector Containing Three Anti-HIV Genes-CCR5 Ribozyme, Tat-rev siRNA, and TAR Decoy-in SCID-hu Mouse-Derived T Cells.

Gene therapeutic strategies show promise in controlling human immunodeficiency virus (HIV) infection and in restoring immunological function. A number of efficacious anti-HIV gene constructs have been described so far, including small interfering RNAs (siRNAs), RNA decoys, transdominant proteins, and ribozymes, each with a different mode of action. However, as HIV is prone to generating escape mutants, the use of a single anti-HIV construct would not be adequate to afford long range-viral protection. On this basis, a combination of highly potent anti-HIV genes-namely, a short hairpin siRNA (shRNA) targeting rev and tat, a transactivation response (TAR) decoy, and a CCR5 ribozyme-have been inserted into a third-generation lentiviral vector. Our recent in vitro studies with this construct, Triple-R, established its efficacy in both T-cell lines and CD34 cell-derived macrophages. In this study, we have evaluated this combinatorial vector in vivo. Vector-transduced CD34 cells were injected into severe combined immunodeficiency (SCID)-hu mouse thy/liv grafts to determine their capacity to give rise to T cells. Our results show that phenotypically normal transgenic T cells are generated that are able to resist HIV-1 infection when challenged in vitro. These important attributes of this combinatorial vector show its promise as an excellent candidate for use in human clinical trials.

RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma.

AIDS patients who develop lymphoma are often treated with transplanted hematopoietic progenitor cells. As a first step in developing a hematopoietic cell-based gene therapy treatment, four patients undergoing treatment with these transplanted cells were also given gene-modified peripheral blood-derived (CD34(+)) hematopoietic progenitor cells expressing three RNA-based anti-HIV moieties (tat/rev short hairpin RNA, TAR decoy, and CCR5 ribozyme). In vitro analysis of these gene-modified cells showed no differences in their hematopoietic potential compared with nontransduced cells. In vitro estimates of successful expression of the anti-HIV moieties were initially as high as 22% but declined to approximately 1% over 4 weeks of culture. Ethical study design required that patients be transplanted with both gene-modified and unmanipulated hematopoietic progenitor cells obtained from the patient by apheresis. Transfected cells were successfully engrafted in all four infused patients by day 11, and there were no unexpected infusion-related toxicities. Persistent vector expression in multiple cell lineages was observed at low levels for up to 24 months, as was expression of the introduced small interfering RNA and ribozyme. Therefore, we have demonstrated stable vector expression in human blood cells after transplantation of autologous gene-modified hematopoietic progenitor cells. These results support the development of an RNA-based cell therapy platform for HIV.

Safety and efficacy of a lentiviral vector containing three anti-HIV genes--CCR5 ribozyme, tat-rev siRNA, and TAR decoy--in SCID-hu mouse-derived T cells.

Gene therapeutic strategies show promise in controlling human immunodeficiency virus (HIV) infection and in restoring immunological function. A number of efficacious anti-HIV gene constructs have been described so far, including small interfering RNAs (siRNAs), RNA decoys, transdominant proteins, and ribozymes, each with a different mode of action. However, as HIV is prone to generating escape mutants, the use of a single anti-HIV construct would not be adequate to afford long range-viral protection. On this basis, a combination of highly potent anti-HIV genes--namely, a short hairpin siRNA (shRNA) targeting rev and tat, a transactivation response (TAR) decoy, and a CCR5 ribozyme--have been inserted into a third-generation lentiviral vector. Our recent in vitro studies with this construct, Triple-R, established its efficacy in both T-cell lines and CD34 cell-derived macrophages. In this study, we have evaluated this combinatorial vector in vivo. Vector-transduced CD34 cells were injected into severe combined immunodeficiency (SCID)-hu mouse thy/liv grafts to determine their capacity to give rise to T cells. Our results show that phenotypically normal transgenic T cells are generated that are able to resist HIV-1 infection when challenged in vitro. These important attributes of this combinatorial vector show its promise as an excellent candidate for use in human clinical trials.

Ex vivo selection and expansion of cells based on expression of a mutated inosine monophosphate dehydrogenase 2 after HIV vector transduction: effects on lymphocytes, monocytes, and CD34+ stem cells.

Hematopoietic progenitor cells (HPCs) represent an ideal target for gene therapy treatment of human immunodeficiency virus (HIV) infection. However, gene delivery into quiescent HPCs by retroviral or lentiviral vectors remains relatively poor. We evaluated a selection scheme based on the expression of a variant of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in the de novo purine biosynthesis pathway. As lymphocytes depend more than other cell types on de novo synthesis of purines, IMPDH inhibitors such as mycophenolic acid (MPA) can selectively expand lymphocytes overexpressing the enzymes. We used HIV vectors to deliver an IMPDH variant into T cells and HPCs. We showed that the transduced T cells became resistant to MPA selection. By expressing a short hairpin RNA gene targeted to the HIV gag transcript, the MPA-selected T cells became resistant to HIV-1 infection. Monocyte/macrophages derived from the transduced HPCs differentiated normally and exhibited normal function as measured by B7 up-regulation and phagocytosis when stimulated. Our results suggest that this system may be applicable as a selection strategy to enrich transduced T lymphocytes and mononuclear cells in vivo for HIV gene therapy.

HIV vector production mediated by Rev protein transduction.

HIV-1-based vectors are promising tools for gene therapy because of their ability to integrate into nondividing cells. Their safety in clinical applications remains a major concern. Recombination events occurring among plasmid constructs during vector production could potentially lead to the generation of replication-competent viruses. The safety of HIV-1-based vectors can be improved by removing all regions of the viral genome that are not absolutely required for vector production or function. In this study, we demonstrate that the HIV-1 rev gene is dispensable for the production of HIV-1-based vectors if the vector-producing cells are supplied with purified Rev protein. We compared the efficiency of vector production among Rev, TAT-Rev (Rev fused to the protein transduction domain of the HIV TAT protein), and Rev/Pep-1 (Rev complexed with the carrier peptide Pep-1). Our results showed that 293T cells efficiently internalized TAT-Rev and Rev/Pep-1 and high-titer vector preparations were obtained with this approach. Vectors generated by such an approach showed little difference in their efficiencies of transduction of established cell lines and primary cells compared with vectors generated by standard plasmid cotransfection. Eliminating the requirement for the HIV-1 rev gene during vector production should improve the safety of applying HIV vectors in human clinical trials.

Design of HIV vectors for efficient gene delivery into human hematopoietic cells.

Vectors derived from human immunodeficiency virus (HIV) hold promise for efficient gene delivery into human hematopoietic cells. In this study, HIV vectors containing different combinations of cis-acting elements, including the HIV central flap sequence, and the woodchuck posttranscriptional regulatory element (WPRE) in combination with two different promoters, were used to transduce primary human lymphocytes and cord blood CD34+ progenitor cells. The effect of these elements on the transduction efficiency and transgene expression was systematically evaluated. The results demonstrate that with the combination of flap, WPRE sequences, and the promoter derived from spleen focus-forming virus (SFFV), a foreign gene can be efficiently delivered into primary human T lymphocytes and cord blood CD34+ cells. The study establishes the parameters for proper vector design to efficiently deliver foreign genes into human hematopoietic cells.

Durability of transgene expression and vector integration: recombinant SV40-derived gene therapy vectors.

Many applications of gene delivery require long-term transgene expression. In dividing cells, this result necessitates vector genome persistence, usually by integrating into cellular DNA. Since recombinant gene delivery vectors derived from tag-deleted, replication-incompetent simian virus-40 (SV40) provide for long-term transgene expression in resting and dividing cells, we tested whether such enduring transgene expression reflected integration into cellular genomes. Several lines of evidence suggested this likelihood. After transduction in vitro, continuously dividing cell lines and continuously stimulated primary cells uniformly showed transgene expression for many months. Mice whose livers were transduced in vivo, partially resected, and allowed to regenerate showed comparable levels of transgene expression in regenerated and preoperative livers. Thus, replicationincompetent SV40 vectors (rSV40) persist in vitro and in vivo despite extensive cell division. We tested the possibility that this persistence reflected integration directly. Southern blot analyses of genomic DNA from transduced 293 cells showed that vector genome incorporation into cell DNA happened within days of transduction. Episomal vector DNA was barely detectable 96 hours post-transduction. Inverted PCR, used to characterize vector integration points, showed vector DNA integrated randomly into the cell genome. The circular rSV40 genome opened at different points in each integrand. A significant proportion of the integrands did not contain the entire vector sequence, but rather only portions thereof. Quantitative Southern blot analysis showed approximately 3.05 transgene copies per cell. Therefore, recombinant SV40 gene delivery vectors integrate into the cellular DNA of both resting and dividing cells, and do so randomly and within days of transduction. This integration may explain long-term transgene expression.