Ingrid Bahner

INHIBITION OF HIV-1 IN HUMAN T-LYMPHOCYTES BY RETROVIRALLY TRANSDUCED ANTI-TAT AND REV HAMMERHEAD RIBOZYMES

Gene therapy for AIDS requires the identification of genes which effectively inhibit HIV-1 replication coupled to an efficient vector system for gene delivery and expression. Hammerhead ribozymes are RNA molecules capable of catalytic cleavage of complementary RNA molecules. Ribozymes targeted against two portions of the HIV-1 genome were designed to cleave HIV RNA in the tat gene (TAT) or in a common exon for tat and rev (TR). The ribozymes were cloned into the LN (LTR-neomycin) retroviral vector plasmids and expressed as part of viral LTR-driven transcripts. The vectors were packaged as amphitropic virions and used to transduce human T-lymphocytes. Expression of the vector transcripts containing the ribozyme sequences was readily detected by Northern blot analysis of the transduced T cells. The T-lymphocytes expressing the anti-HIV-1 ribozymes showed resistance to HIV-1 replication. In contrast, cells expressing mutant ribozymes, containing substitutions of a key nucleotide in the catalytic domain which cripples the cleavage activity of the ribozymes, supported replication of HIV-1, demonstrating that the functional ribozymes were cleaving the target RNAs. These studies demonstrate that retrovirally transduced ribozymes included in long, multifunctional transcripts, can inhibit HIV replication in human T-lymphocytes. The ribozyme and expression strategies described here should be useful for the gene therapy of AIDS by conferring resistance to HIV-1 replication on cells derived from transduced hematopoietic stem cells.

Novel Pol II Fusion Promoter Directs Human Immunodeficiency Virus Type 1-Inducible Coexpression of a Short Hairpin RNA and Protein

ABSTRACT We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.

Retroviral-mediated transfer of the human glucocerebrosidase gene into cultured Gaucher bone marrow.

Gaucher disease, a lysosomal glycolipid storage disorder, results from the genetic deficiency of an acidic glucosidase, glucocerebrosidase (GC). The beneficial effects of allogeneic bone marrow transplantation (BMT) for Gaucher disease suggest that GC gene transduction and the transplantation of autologous hematopoietic stem cells (gene therapy) may similarly alleviate symptoms. We have constructed a retroviral vector, L-GC, produced by a clone of the amphotropic packaging cell line PA317, which transduces the normal human GC cDNA with high efficiency. Whole-marrow mononuclear cells and CD34-enriched cells from a 4-yr-old female with type 3 Gaucher disease were transduced by the L-GC vector and studied in long-term bone marrow culture (LTBMC). Prestimulation of marrow with IL-3 and IL-6, followed by co-cultivation with vector-producing fibroblasts, produced gene transfer into 40-45% of the hematopoietic progenitor cells. The levels of GC expression in progeny cells (primarily mature myelomonocytic) produced by the LTBMC were quantitatively analyzed by Northern blot, Western blot, and glucocerebrosidase enzyme assay. Normal levels of GC RNA, immunoreactive protein, and enzymatic activity were detected throughout the duration of culture. These studies demonstrate that retroviral vectors can efficiently transfer the GC gene into long-lived hematopoietic progenitor cells from the bone marrow of patients with Gaucher disease and express physiologically relevant levels of GC enzyme activity.

Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34(+) HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8(+) and CD4(+) T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4-5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8(+) and CD4(+) T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34(+) HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.

Gene transfer into human umbilical cord blood-derived CD34+ cells by particle-mediated gene transfer.

Delivery of genes into hematopoietic progenitor cells offers an attractive means for the introduction of corrective or protective genes into cells of both the myeloid and lymphoid lineage. Previously, investigators have often used murine retroviral vectors for gene delivery which require cells to be cycling for efficient delivery. We describe a nonviral method of gene delivery using particle-mediated gene transfer to obviate many disadvantages of viral vectors related to safety, production costs and the need for cell cycle proliferation. Using a CMV-CAT reporter plasmid, we show transfection of highly purified CD34+ cells isolated from umbilical cord blood. Effective gene transfer was shown in unstimulated and in growth-stimulated cells. Fol- lowing transfection with a neomycin resistance gene, differentiation into cells of the myeloid lineage was observed, assayed by CFU-GM in the presence of G-418. Both unstimulated and stimulated cells gave rise to CFU-GM in the presence of G-418, indicating that stable expression of the neomycin resistance gene was maintained in early progenitors. These results demonstrate that particle-mediated gene transfer into human hematopoietic cells from umbilical cord blood can be achieved without affecting their CFU-GM differentiation potential. This gene transfer method offers an alternative approach to gene therapy studies involving human hematopoietic progenitor cells.

Teaching Science through Biomedical Research in an Elective Curriculum

The Scholarly Concentrations Program (SCP) at the USF Health Morsani College of Medicine (MCOM) was initiated in 2007 to foster the intellectual growth of its students within the larger context of medicine beyond the core medical curriculum1. Specifically, the students participate in a longitudinal curriculum and are mentored to participate in areas of inquiry related to their concentration focus. The research Scholarly Concentration (rSC) is one of ten different scholarly concentrations: Innovation, Entrepreneurship and Business, Health Disparities, Health Systems Engineering, International Medicine, Law and Medicine, Medical Education, Medicine and Gender, Medical Humanities, Public Health and Research. Although the SCP is an elective program, student enrollment has consistently increased since its inception and now comprises > 90% of the class of 2015. Here we report and discuss the outcome data of the rSC as a model to teach basic research skills and expand the study of biomedical sciences during undergraduate medical education.

HIV-1 gp41 ectodomain enhances Cryptococcus neoformans binding to human brain microvascular endothelial cells via gp41 core-induced membrane activities

Cryptococcus neoformans causes life-threatening meningoencephalitis, particularly prevalent in AIDS patients. The interrelationship between C. neoformans and HIV-1 is intriguing, as both pathogens elicit severe neuropathological complications. We have previously demonstrated that the HIV-1 gp41 ectodomain fragments gp41-I33 (amino acids 579-611) and gp41-I90 (amino acids 550-639) can enhance C. neoformans binding to HBMECs (human brain microvascular endothelial cells). Both peptides contain the loop region of gp41. In the present study, we used immunofluorescence microscopy and transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 (intercellular adhesion molecule 1) on the HBMECs and elicited membrane ruffling on the surface of HBMECs. The HIV-1 gp41 ectodomain could also induce CD44 and β-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα (protein kinase Cα) phosphorylation like C. neoformans. Instead, gp41-I90 was able to induce syncytium formation on HBMECs. The results of the present study suggest HIV-1 gp41-enhanced C. neoformans binding to HBMECs via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.

998. The HIV-1 LTR-hsp70 Fusion Promoter Directs HIV-1 Inducible Expression of Anti-Rev siRNA along with HIV-1 Rev Transdominant Mutant RevM10

Top of pageAbstract Small interfering RNA (siRNA)-mediated knockdown of deleterious transcripts of cellular or viral origin has potential applications for treatment of hereditary diseases, infections and cancer. We describe here a method to further supplement the potency of siRNA by simultaneous delivery of a gene in conjunction with an siRNA from a novel promoter cassette. This technique can be applied to deliver corrective genes and simultaneously knock out deleterious gene expression. The gene in question can be a corrected replacement gene with codon modifications to prevent the siRNA from targeting the corrective gene, in the case of hereditary diseases, or a suicide/transdominant mutant gene employed to further potentiate the anti-tumor or anti-viral effects of siRNA. As proof of principle we have expressed the HIV-1 transdominant mutant revM10 in conjunction with an anti-Rev siRNA from our HIV-1 LTR, Drosophila hsp70 fusion promoter to further potentiate the anti-HIV-1 effect of siRNA and also to offset resistant mutants that may arise from any single approach. HIV-1 inducible siRNA expression or RevM10 expression is observed only when cells are infected with pNL4-3. A potent synergistic response is observed in cells transfected with siRNA-RevM10 construct as compared with cells transfected with siRNA alone. We are in the process of monitoring whether or not the dual cassette reduces the frequency of viral mutants resistant to one or the other antiviral agent when applied alone. The potential applications of this approach in gene therapy settings will be discussed.

987. Foamy Virus Vectors That Block HIV Infection

Foamy virus (FV) vectors have been demonstrated to transduce human hematopoietic stem cells with a high frequency. We have developed FV vectors that can block HIV infection with the intention of developing a therapeutic vector. Additional features of FV vectors that support their use in clinical gene therapy include: a) no evidence of disease from the prototypic FV in infected humans and in non-human primates, b) reduced recombination potential with HIV in infected patients, c) FV vectors are self-inactivating vectors, and d) relative to lentivirus vectors, FV vector production does not share molecular pathways with HIV.