Humanized Rag1-/- γc-/- mice support multilineage hematopoiesis and are susceptible to HIV-1 infection via systemic and vaginal routes.

Several new immunodeficient mouse models for human cell engraftment have recently been introduced that include the Rag2(-/-)γc(-/-), NOD/SCID, NOD/SCIDγc(-/-) and NOD/SCIDß2m(-/-) strains. Transplantation of these mice with CD34(+) human hematopoietic stem cells leads to prolonged engraftment, multilineage hematopoiesis and the capacity to generate human immune responses against a variety of antigens. However, the various mouse strains used and different methods of engrafting human cells are beginning to illustrate strain specific variations in engraftment levels, duration and longevity of mouse life span. In these proof-of-concept studies we evaluated the Balb/c-Rag1(-/-)γ(-/-) strain for engraftment by human fetal liver derived CD34(+) hematopoietic cells using the same protocol found to be effective for Balb/c-Rag2(-/-)γc(-/-) mice. We demonstrate that these mice can be efficiently engrafted and show multilineage human hematopoiesis with human cells populating different lymphoid organs. Generation of human cells continues beyond a year and production of human immunoglobulins is noted. Infection with HIV-1 leads to chronic viremia with a resultant CD4 T cell loss. To mimic the predominant sexual viral transmission, we challenged humanized Rag1(-/-)γc(-/-) mice with HIV-1 via vaginal route which also resulted in chronic viremia and helper T cell loss. Thus these mice can be further exploited for studying human pathogens that infect the human hematopoietic system in an in vivo setting.

An aptamer-siRNA chimera suppresses HIV-1 viral loads and protects from helper CD4(+) T cell decline in humanized mice.

Therapeutic strategies designed to treat HIV infection with combinations of antiviral drugs have proven to be the best approach for slowing the progression to AIDS. Despite this progress, there are problems with viral drug resistance and toxicity, necessitating new approaches to combating HIV-1 infection. We have therefore developed a different combination approach for the treatment of HIV infection in which an RNA aptamer, with high binding affinity to the HIV-1 envelope (gp120) protein and virus neutralization properties, is attached to and delivers a small interfering RNA (siRNA) that triggers sequence-specific degradation of HIV RNAs. We have tested the antiviral activities of these chimeric RNAs in a humanized Rag2(-/-)γc(-/-) (RAG-hu) mouse model with multilineage human hematopoiesis. In this animal model, HIV-1 replication and CD4(+) T cell depletion mimic the situation seen in human HIV-infected patients. Our results show that treatment with either the anti-gp120 aptamer or the aptamer-siRNA chimera suppressed HIV-1 replication by several orders of magnitude and prevented the viral-induced helper CD4(+) T cell decline. In comparison to the aptamer alone, the aptamer-siRNA combination provided more extensive inhibition, resulting in a significantly longer antiviral effect that extended several weeks beyond the last injected dose. The aptamer thus acts as a broad-spectrum HIV-neutralizing agent and an siRNA delivery vehicle. The combined aptamer-siRNA agent provides an attractive, nontoxic therapeutic approach for treatment of HIV infection.

Humanized Rag2(-/-)gammac(-/-) (RAG-hu) mice can sustain long-term chronic HIV-1 infection lasting more than a year.

HIV-1 infection is characterized by life-long viral persistence and continued decline of helper CD4 T cells. The new generation of humanized mouse models that encompass RAG-hu, hNOG and BLT mice have been shown to be susceptible to HIV-1 infection and display CD4 T cell loss. Productive infection has been demonstrated with both R5 and X4 tropic strains of HIV-1 via direct injection as well as mucosal exposure. However the duration of infection in these mice was evaluated for a limited time lasting only weeks post infection, and it is not established how long the viremia can be sustained, and if the CD4 T cell loss persists throughout the life of the infected humanized mice. In the present study we followed the HIV-1 infected RAG-hu mice to determine the long-term viral persistence and CD4 T cell levels. Our results showed that viremia persists life-long lasting for more than a year, and that CD4 T cell levels display a continuous declining trend as seen in the human. These studies provide a chronic HIV-1 infection humanized mouse model that can be used to dissect viral latency, long-term drug evaluation and immune-based therapies.

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.

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.

Lentiviral transduction of Tar Decoy and CCR5 ribozyme into CD34+ progenitor cells and derivation of HIV-1 resistant T cells and macrophages.

BACKGROUND: RNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy. These include ribozymes, aptamers (decoys), and small interfering RNAs (siRNAs). Lentiviral vectors are ideal for transduction of such inhibitory RNAs into hematopoietic stem cells due to their ability to transduce non-dividing cells and their relative refractiveness to gene silencing. The objective of this study is to introduce an HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme into CD34+ hematopoietic progenitor cells via an HIV-based lentiviral vector to derive viral resistant progeny T cells and macrophages. RESULTS: High efficiency and sustained gene transfer into CD34+ cells were achieved with lentiviral vector constructs harboring either Tar decoy or Tar decoy in combination with CCR5 ribozyme. Cells transduced with these constructs differentiated normally into T-lymphocytes in vivo in thy/liv grafts of SCID-hu mice, and into macrophages in vitro in the presence of appropriate growth factors. When challenged in vitro, the differentiated T lymphocytes and macrophages showed marked resistance against HIV-1 infection. CONCLUSIONS: Viral resistant transgenic T cells and macrophages that express HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme could be obtained by lentiviral gene transduction of CD34+ progenitor cells. These results showed for the first time that expression of these anti-HIV-1 transgenes in combination do not interfere with normal thymopoiesis and thus have set the stage for their application in stem cell based gene therapy for HIV/AIDS.

Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in T lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages.

The phenomenon of RNA interference mediated by small interfering RNAs (siRNAs) is a potent gene-silencing mechanism. A number of recent studies demonstrated inhibition of HIV-1 replication in cultured cells using this approach. To make further progress and harness this technology for HIV-1 gene therapy in a stem cell setting, in vivo studies using primary hematopoietic cells are needed. Using an HIV-based lentiviral vector we introduced an anti-Rev siRNA construct into CD34(+) hematopoietic progenitor cells. The siRNA-transduced progenitor cells were allowed to mature into macrophages in vitro and T cells in vivo in SCID-hu mouse thy/liv grafts. Phenotypically normal T cells and macrophages displaying characteristic surface markers were obtained. In vitro HIV-1 challenge of the siRNA-expressing macrophages and T cells with macrophage-tropic and T-cell-tropic HIV-1, respectively, showed marked viral resistance. These experiments demonstrate the utility of siRNAs delivered into hematopoietic stem cells via lentiviral vectors for future in vivo applications.