HIV Gene Therapy Research Articles

57. Optimized Lentiviral Vectors for HIV Gene Therapy: Multiplexed Expression of Small RNAs and Inclusion of MGMTP140K Drug Resistance Gene

57. Optimized Lentiviral Vectors for HIV Gene Therapy: Multiplexed Expression of Small RNAs and Inclusion of MGMTP140K Drug Resistance Gene

Silencing of HIV-1 gag gene from epidemic strains among men who have sex with men (MSM) in Tianjin, China by a broad-spectrum short hairpin RNA.

RNA interference (RNAi) has been successfully used as a promising method to inhibit the replication of different viruses, including human immunodeficiency virus (HIV). Gene mutation is a hurdle for the anti-HIV by RNAi. Although prone to mutation, some genes are conserved and limited in functionally important regions. The gag gene is conserved in different subtypes and plays an important role in the assembly of HIV viral particle. Here, we identified subtypes and conserved sequences within forty-four gag genes from the epidemic strains among men who have sex with men. Three subtypes of gag gene, including CRF01_AE (47.7%), CRF07_BC (40.9%) and B (11.4%) were analyzed by online blast. We designed five small hairpin RNAs (shRNAs) based on the conserved sequences. The gag-EGFP fusion transcript reporter system was used to select the most efficient shRNA. Among the five candidate shRNAs, gag-shRNA-3 represented a broad-spectrum inhibition against all chosen targets. This broad-spectrum shRNA diminished the titer of subtypes B and C of HIV-1 for a hundred orders of magnitude. The gag-shRNA-3 described here is a potential therapeutic agent in the HIV-1 gene therapy.

Gene therapy for HIV?

Gene therapy for HIV?

An Efficient Large-Scale Retroviral Transduction Method Involving Preloading the Vector into a RetroNectin-Coated Bag with Low-Temperature Shaking

In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines. To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel (i.e., the RetroNectin-bound virus transduction method). In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system. Retroviral vector was preloaded into a RetroNectin-coated bag and incubated at 4°C for 16 h on a reciprocating shaker at 50 rounds per minute. After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag. Finally, we performed validation runs of endoribonuclease MazF-modified CD4+ T cell manufacturing for HIV-1 gene therapy and T cell receptor-modified T cell manufacturing for MAGE-A4 antigen-expressing cancer gene therapy and achieved over 200-fold (≥1010) and 100-fold (≥5×109) expansion, respectively. In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols.

CD4(+) T Cells Modified by the Endoribonuclease MazF Are Safe and Can Persist in SHIV-infected Rhesus Macaques.

MazF, an endoribonuclease encoded by Escherichia coli, specifically cleaves the ACA (adenine–cytosine–adenine) sequence of single-stranded RNAs. Conditional expression of MazF under the control of the HIV-1 LTR promoter rendered CD4+ T cells resistant to HIV-1 replication without affecting cell growth. To investigate the safety, persistence and efficacy of MazF-modified CD4+ T cells in a nonhuman primate model in vivo, rhesus macaques were infected with a pathogenic simian/human immunodeficiency virus (SHIV) and transplanted with autologous MazF-modified CD4+ T cells. MazF-modified CD4+ T cells were clearly detected throughout the experimental period of more than 6 months. The CD4+ T cell count values increased in all four rhesus macaques. Moreover, the transplantation of the MazF-modified CD4+ T cells was not immunogenic, and did not elicit cellular or humoral immune responses. These data suggest that the autologous transplantation of MazF-modified CD4+ T cells in the presence of SHIV is effective, safe and not immunogenic, indicating that this is an attractive strategy for HIV-1 gene therapy.

CCR5 Gene Editing of Resting CD4(+) T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice.

CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4+ T cells. Both activated and resting primary CD4+ T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4+ T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2rγc null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN–transduced resting CD4+ T cells from treatment naïve as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy.

Stem-Cell-Based Gene Therapy for HIV Infection

Despite the enormous success of combined anti-retroviral therapy, HIV infection is still a lifelong disease and continues to spread rapidly worldwide. There is a pressing need to develop a treatment that will cure HIV infection. Recent progress in stem cell manipulation and advancements in humanized mouse models have allowed rapid developments of gene therapy for HIV treatment. In this review, we will discuss two aspects of HIV gene therapy using human hematopoietic stem cells. The first is to generate immune systems resistant to HIV infection while the second strategy involves enhancing anti-HIV immunity to eliminate HIV infected cells.

Foamy virus vectors for HIV gene therapy.

Highly active antiretroviral therapy (HAART) has vastly improved outcomes for patients infected with HIV, yet it is a lifelong regimen that is expensive and has significant side effects. Retroviral gene therapy is a promising alternative treatment for HIV/AIDS; however, inefficient gene delivery to hematopoietic stem cells (HSCs) has so far limited the efficacy of this approach. Foamy virus (FV) vectors are derived from non-pathogenic viruses that are not endemic to the human population. FV vectors have been used to deliver HIV-inhibiting transgenes to human HSCs, and they have several advantages relative to other retroviral vectors. These include an attractive safety profile, broad tropism, a large transgene capacity, and the ability to persist in quiescent cells. In addition, the titers of FV vectors are not reduced by anti-HIV transgenes that affect the production of lentivirus (LV) vectors. Thus FV vectors are very promising for anti-HIV gene therapy. This review covers the advantages of FV vectors and describes their preclinical development for anti-HIV gene therapy.

Revisiting Transdominant-Negative Proteins in HIV Gene Therapy

HIV remains a global public health issue and new therapies are actively being developed. Traditional treatments such as small-molecule inhibitors are being investigated; however, newer modalities are also being pursued, including the use of transdominant-negative proteins. A transdominant negative is a mutant of a protein designed to interfere with the normal activity of its wild-type counterpart. Transdominant negatives designed to block HIV replication are based on viral proteins; however, recent approaches show that transdominant negatives of cellular proteins have therapeutic potential. Recent discoveries have revealed that treatments based on transdominant negatives can greatly disrupt the replication cycle of the virus. This article aims to review viral and cellular protein-based transdominant negatives, the recent elucidation of their modes of action and their potential use in HIV gene therapy.

Combinatorial RNA-based gene therapy for the treatment of HIV/AIDS

Introduction: HIV/AIDS continues to be a worldwide health problem and viral eradication has been an elusive goal. HIV+ patients are currently treated with combination antiretroviral therapy (cART) which is not curative. For many patients, cART is inaccessible, intolerable or unaffordable. Therefore, a new class of therapeutics for HIV is required to overcome these limitations. Cell and gene therapy for HIV has been proposed as a way to provide a functional cure for HIV in the form of a virus/infection resistant immune system.Areas covered: In this review, the authors describe the standard therapy for HIV/AIDS, its limitations, current areas of investigation and the potential of hematopoietic stem cells modified with anti-HIV RNAs as a means to affect a functional cure for HIV.Expert opinion: Cell and gene therapy for HIV/AIDS is a promising alternative to antiviral drug therapy and may provide a functional cure. In order to show clinical benefit, multiple mechanisms of inhibition of HIV entry and lifecycle are likely to be required. Among the most promising antiviral strategies is the use of transgenic RNA molecules that provide protection from HIV infection. When these molecules are delivered as gene-modified hematopoietic stem and progenitor cells, long-term repopulation of the patient's immune system with gene-modified progeny has been observed.

Whiskers-less HIV-protease: a possible way for HIV-1 deactivation.

BackgroundAmong viral enzymes, the human HIV-1 protease comprises the most interesting target for drug discovery. There are increasing efforts focused on designing more effective inhibitors for HIV-1 protease in order to prevent viral replication in AIDS patients. The frequent and continuous mutation of HIV-1 protease gene creates a formidable obstacle for enzyme inhibition which could not be overcome by the traditional single drug therapy. Nowadays, in vitro and in silico studies of protease inhibition constitute an advanced field in biological researches. In this article, we tried to simulate protease-substrate complexes in different states; a native state and states with whiskers deleted from one and two subunits. Molecular dynamic simulations were carried out in a cubic box filled with explicit water at 37°C and in 1atomsphere of pressure.ResultsOur results showed that whisker truncation of protease subunits causes the dimer structure to decrease in compactness, disrupts substrate-binding site interactions and changes in flap status simultaneously.ConclusionsBased on our findings we claim that whisker truncation even when applied to a single subunit, threats dimer association which probably leads to enzyme inactivation. We may postulate that inserting a gene to express truncated protease inside infected cells can interfere with protease dimerization. The resulted proteases would presumably have a combination of native and truncated subunits in their structures which exert no enzyme activities as evidenced by the present work. Our finding may create a new field of research in HIV gene therapy for protease inhibition, circumventing problems of drug resistance.

CD25 Preselective Anti-HIV Vectors for Improved HIV Gene Therapy

As HIV continues to be a global public health problem with no effective vaccine available, new and innovative therapies, including HIV gene therapies, need to be developed. Due to low transduction efficiencies that lead to low in vivo gene marking, therapeutically relevant efficacy of HIV gene therapy has been difficult to achieve in a clinical setting. Methods to improve the transplantation of enriched populations of anti-HIV vector-transduced cells may greatly increase the in vivo efficacy of HIV gene therapies. Here we describe the development of preselective anti-HIV lentiviral vectors that allow for the purification of vector-transduced cells to achieve an enriched population of HIV-resistant cells. A selectable protein, human CD25, not normally found on CD34+ hematopoietic progenitor cells (HPCs), was incorporated into a triple combination anti-HIV lentiviral vector. Upon purification of cells transduced with the preselective anti-HIV vector, safety was demonstrated in CD34+ HPCs and in HPC-derived macrophages in vitro. Upon challenge with HIV-1, improved efficacy was observed in purified preselective anti-HIV vector-transduced macrophages compared to unpurified cells. These proof-of-concept results highlight the potential use of this method to improve HIV stem cell gene therapy for future clinical applications.

Endogenous MCM7 MicroRNA Cluster as a Novel Platform to Multiplex Small Interfering and Nucleolar RNAs for Combinational HIV-1 Gene Therapy

Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.

Zinc finger protein designed to target 2-long terminal repeat junctions interferes with human immunodeficiency virus integration.

Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine-adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future.

344. Optimization of the MCM7 miRNA Cluster as a Novel Platform to Multiplex Small Interfering and Nucleolar RNAs for Combinational HIV-1 Gene Therapy

344. Optimization of the MCM7 miRNA Cluster as a Novel Platform to Multiplex Small Interfering and Nucleolar RNAs for Combinational HIV-1 Gene Therapy

Lens Epithelium-derived Growth Factor/p75 Qualifies as a Target for HIV Gene Therapy in the NSG Mouse Model

Lens epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF(325-530)) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD(4)(+) T-cells were transduced with lentiviral vectors encoding LEDGF(325-530), the interaction-deficient mutant LEDGF(325-530)D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF(325-530) overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD(4)(+) T-cells expressing LEDGF(325-530) in comparison with engraftment with LEDGF(325-530)D366N cells. Four weeks after transplantation with LEDGF(325-530)D366N cells, 70% of the CD(4)(+) cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF(325-530) cells only a 20% decrease in CD(4)(+) cells was measured. Liver and spleen sections of LEDGF(325-530) mice contained less HIV than LEDGF(325-530)D366N mice as measured by p24 antigen detection. LEDGF(325-530) overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD(4)(+) T-cells.

AAV6-mediated Systemic shRNA Delivery Reverses Disease in a Mouse Model of Facioscapulohumeral Muscular Dystrophy

Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans.

Current Progress and Challenges in HIV Gene Therapy

HIV-1 causes AIDS, a syndrome that affects millions of people globally. Existing HAART is efficient in slowing down disease progression but cannot eradicate the virus. Furthermore the severity of the side effects and the emergence of drug-resistant mutants call for better therapy. Gene therapy serves as an attractive alternative as it reconstitutes the immune system with HIV-resistant cells and could thereby provide a potential cure. The feasibility of this approach was first demonstrated with the 'Berlin patient', who was functionally cured from HIV/AIDS with undetectable HIV-1 viral load after transplantation of bone marrow harboring a naturally occurring CCR5 mutation that blocks viral entry. Here, we give an overview of the current status of HIV gene therapy and remaining challenges and obstacles.

Antiviral restriction factor transgenesis in the domestic cat

Studies of the domestic cat have contributed to many scientific advances, including the present understanding of the mammalian cerebral cortex. A practical capability for cat transgenesis is needed to realize the distinctive potential of research on this neurobehaviorally complex, accessible species for advancing human and feline health. For example, humans and cats are afflicted with pandemic AIDS lentiviruses that are susceptible to species-specific restriction factors. Here we introduced genes encoding such a factor, rhesus macaque TRIMCyp, and eGFP, into the cat germline. The method establishes gamete-targeted transgenesis for the first time in a carnivore. We observed uniformly transgenic outcomes, widespread expression, no mosaicism and no F1 silencing. TRIMCyp transgenic cat lymphocytes resisted feline immunodeficiency virus replication. This capability to experimentally manipulate the genome of an AIDS-susceptible species can be used to test the potential of restriction factors for HIV gene therapy and to build models of other infectious and noninfectious diseases.

In Vivo Safety and Persistence of Endoribonuclease Gene-Transduced CD4+ T Cells in Cynomolgus Macaques for HIV-1 Gene Therapy Model

BackgroundMazF is an endoribonuclease encoded by Escherichia coli that specifically cleaves the ACA sequence of mRNA. In our previous report, conditional expression of MazF in the HIV-1 LTR rendered CD4+ T lymphocytes resistant to HIV-1 replication. In this study, we examined the in vivo safety and persistence of MazF-transduced cynomolgus macaque CD4+ T cells infused into autologous monkeys.Methodology/Principal FindingsThe in vivo persistence of the gene-modified CD4+ T cells in the peripheral blood was monitored for more than half a year using quantitative real-time PCR and flow cytometry, followed by experimental autopsy in order to examine the safety and distribution pattern of the infused cells in several organs. Although the levels of the MazF-transduced CD4+ T cells gradually decreased in the peripheral blood, they were clearly detected throughout the experimental period. Moreover, the infused cells were detected in the distal lymphoid tissues, such as several lymph nodes and the spleen. Histopathological analyses of tissues revealed that there were no lesions related to the infused gene modified cells. Antibodies against MazF were not detected. These data suggest the safety and the low immunogenicity of MazF-transduced CD4+ T cells. Finally, gene modified cells harvested from the monkey more than half a year post-infusion suppressed the replication of SHIV 89.6P.Conclusions/SignificanceThe long-term persistence, safety and continuous HIV replication resistance of the mazF gene-modified CD4+ T cells in the non-human primate model suggests that autologous transplantation of mazF gene-modified cells is an attractive strategy for HIV gene therapy.