Shirley Li

RNA-Based Gene Therapy for HIV with Lentiviral Vector–Modified CD34+ Cells in Patients Undergoing Transplantation for AIDS-Related Lymphoma

Transfected stem cells transplanted into patients with HIV infection resulted in sustained RNA expression of introduced genes in blood cells for up to 2 years. Steps Toward a Stable Source of Therapeutic RNA Gene therapy in humans has not been easy to implement. Genes inserted into complex human cells have triggered serious unintended consequences and have often proven to be short-lived. Yet perseverance may be paying off. DiGiusto et al. report a step toward workable gene therapy in the form of stable expression of a lentiviral vector encoding anti-HIV RNAs in blood stem cells transplanted into AIDS patients. None of these patients is cured, but the vector seems to stably express the potentially therapeutic RNAs. Putting exogenous gene sequences into humans is risky, and review boards are appropriately conservative. But DiGiusto et al. took advantage of a clinical situation to design a trial that minimized extra risk to the subjects. Blood cancer (lymphoma) is common in AIDS patients, and they are often treated by ablation of their diseased bone marrow with chemotherapy followed by a transplant with their own previously saved blood stem cells. Because these patients were being transplanted with their own blood cells anyway, the authors were able to get permission to transfect a few of the blood cells of four patients with a vector carrying anti-HIV entities and reinfuse them along with the normally transplanted cells. The vector made RNAs that could counteract viral replication in several ways: inhibition of viral entry (with a CCR5 ribozyme), inhibition of RNA transport [by a small interfering RNA (siRNA) to tat/rev], and inhibition of viral transcription initiation with a decoy RNA. The good news was that the patients showed no signs of toxicity besides problems usually associated with transplantation and that blood cells from all four patients contained signs of the transplanted genes, with the amounts increasing in two of the patients after 18 months. Although the fraction of cells containing the genes was <0.2%, this was not too different from the fraction of transfected cells that was infused into the patients. The three anti-HIV RNAs could also be detected as long as 1 year after the initial infusion, and examination of T cells, monocytes, and B cells from one patient confirmed the presence of vector in these three cell types. These cells that survived for long periods of time in patients, although too scarce to cure or even improve their HIV infections, nevertheless offer lessons for future applications of gene therapy. We know that this procedure is seemingly safe and that cells given new genetic material via a lentiviral vector outside the patient can survive once reimplanted. Continued perseverance can only bring us closer to realizing the potential of this promising therapy. 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 ~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.

Expression of small, therapeutic RNAs in human cell nuclei

Effective intracellular expression of small RNA therapeutics depends on a number of factors. The RNA, whether antisense, ribozyme, or RNA aptamer, must be efficiently transcribed, stabilized against rapid degradation, folded correctly, and directed to the part of the cell where it can be most effective. To overcome a number of these problems we have been testing expression cassettes based on the human tRNAmet and U6 snRNA promoters, in which transcripts encoding small RNA inserts are protected against attack from the 3′ end. Transient expression in cultured cells results in 103–2 × 107 full-length transcripts per cell, depending partially on the promoter construct used but also on the nature of the insert RNA. 5′ γ-Phosphate methylation (capping) depended, as expected, on the inclusion of specific U6 snRNA sequences from positions +19 to +27. In situ localization of the transcripts shows that both tRNA and U6 promoter transcripts give primarily punctate nuclear patterns, and that capping of transcripts is not required for nuclear retention. Several different insert RNAs directed against HIV-1 were tested by cotransfection with HIV-1 provirus and assay for subsequent viral reverse transcriptase production. These include antisense RNA, hairpin and hammerhead ribozymes, and RNA ligands (aptamers) for Tat and Rev RNA binding proteins. Results show that Rev-binding RNAs efficiently block HIV-1 gene expression, whereas other RNAs have little or no effect when expressed in these cassettes.

Novel Dual Inhibitory Function Aptamer–siRNA Delivery System for HIV-1 Therapy

The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. In this study, we demonstrate cell type-specific delivery of anti-human immunodeficiency virus (anti-HIV) siRNAs through fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1-infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and that the appended siRNA is processed by Dicer; this releases an anti-tat/rev siRNA which, in turn, inhibits HIV replication. We show for the first time a dual functioning aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also show that gp120 expressed on the surface of HIV-infected cells can be used for aptamer-mediated delivery of anti-HIV siRNAs.

Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs.

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.

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.

A nucleolar TAR decoy inhibitor of HIV-1 replication

Tat is a critical regulatory factor in HIV-1 gene expression. It mediates the transactivation of transcription from the HIV-1 LTR by binding to the transactivation response (TAR) element in a complex with cyclin T1. Because of its critical and early role in HIV gene expression, Tat and its interaction with the TAR element constitute important therapeutic targets for the treatment of HIV-1 infection. Based on the known nucleolar localization properties of Tat, we constructed a chimeric small nucleolar RNA-TAR decoy that localizes to the nucleoli of human cells and colocalizes in the nucleolus with a Tat-enhanced GFP fusion protein. When the chimeric RNA was stably expressed in human T lymphoblastoid CEM cells it potently inhibited HIV-1 replication. These results demonstrate that the nucleolar trafficking of Tat is critical for HIV-1 replication and suggests a role for the nucleolus in HIV-1 viral replication.

RNA‐Mediated Inhibition of HIV in a Gene Therapy Setting

Abstract: At present, treatment for HIV‐1 infection employs highly active anti‐retroviral therapy (HAART), which utilizes a combination of RT and protease inhibitors. Unfortunately, HIV can escape many therapies because of its high mutation rate and the complexity of its pathogenesis. HIV‐1 integrates into the cellular genome, which facilitates persistence and acts as a reservoir for reactivation and replication. As an alternative or adjuvant to chemotherapy we have been developing an RNA‐based gene therapy approach for the treatment of HIV‐1 infection. This article summarizes the various RNA based technologies that we have developed for potential application in a gene therapy setting.

Localized expression of small RNA inhibitors in human cells

Several types of small RNAs have been proposed as gene expression repressors with great potential for use in gene therapy. RNA polymerase III (pol III) provides an ideal means of expressing small RNAs in cells because its normal products are small, highly structured RNAs that are found in a variety of subcellular compartments. We have designed cassettes that use human pol III promoters for the high-level expression of small RNAs in the cytoplasm, nucleoplasm, and nucleolus. The levels and subcellular destinations of the transcripts are compared for transcripts expressed using the U6 small nuclear RNA (snRNA), 5S ribosomal RNA (rRNA), and the 7SL RNA component of the signal recognition particle. The most effective location for a particular inhibitory RNA is not necessarily predictable; thus these cassettes allow testing of the same RNA insert in multiple subcellular locations. Several small interfering RNA (siRNA) inserts were tested for efficacy. An siRNA insert that reduces lamin expression when transcribed from the U6 snRNA promoter in the nucleus has no effect on lamin expression when transcribed from 5S rRNA and 7SL RNA-based cassettes and found in the nucleolus and cytoplasm. To test further the generality of U6-driven siRNA inhibitors, siRNAs targeting HIV were tested by co-transfection with provirus in cell culture. Although the degree of HIV-1 inhibition varied among inserts, results show that the U6 cassette provides a means of expressing an siRNA-like inhibitor of HIV gene expression.

Plasma viremia and cellular HIV-1 DNA persist despite autologous hematopoietic stem cell transplantation for HIV-related lymphoma.

Abstract: A cure of HIV-1 has been achieved in one individual through allogeneic stem cell transplantation with a CCR5[INCREMENT]32 homozygous donor. Whether myeloablation and autologous stem cell transplantation for lymphoma in patients on suppressive antiretroviral therapy can eliminate HIV-1 reservoirs is unknown. Low-level plasma viremia and total HIV-1 DNA and 2-LTR circles in blood mononuclear cells were quantified after autologous transplantation in 10 patients on suppressive antiretroviral therapy using quantitative polymerase chain reaction assays capable of single-copy nucleic acid detection. Plasma viremia was detectable in 9 patients, whereas HIV-1 DNA was detectable in all 10 patients, indicating that HIV-1 had not been eliminated.

Inhibition of HIV-1 replication with designed miRNAs expressed from RNA polymerase II promoters

Small interfering RNA (siRNA) mediates sequence-specific RNA cleavage and represents a potential approach to treat the infection of human immunodeficiency virus (HIV). Expression of a single siRNA species frequently led to the emergence of HIV escape variants. Thus, multiple siRNAs targeted to different regions in the HIV-1 genome may be required. However, overexpression of different anti-HIV siRNA genes from multiple pol III promoters can induce cell toxicity, thus may not be a viable option in the setting of human gene therapy trials. In the current study, we evaluated the strategy of using pol II promoters to drive the expression of siRNAs against HIV-1. We replaced the stem sequence in the stem-loop structure of the well-characterized miR-30a with siRNA sequences and showed that designed microRNA (miRNA) could be expressed from pol II promoters. We demonstrated efficient inhibition of HIV-1 replication with such designed miRNA, but the efficacy was directly correlated with the expression level. Both the vector copy number and the promoter strength directly affected the ability of the siRNA to inhibit HIV-1 replication. We also showed that a combination of pol II and pol III promoters to express two different siRNAs increased the efficacy against HIV-1 replication without comprising cell viability.