Platform for isolation and characterization of SARS-CoV-2 variants enables rapid characterization of Omicron in Australia.

Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021.

The feasibility of incorporating Vpx into lentiviral gene therapy vectors.

While current antiretroviral therapy has significantly improved, challenges still remain in life-long targeting of HIV-1 reservoirs. Lentiviral gene therapy has the potential to deliver protective genes into the HIV-1 reservoir. However, inefficient reverse transcription (RT) occurs in HIV-1 reservoirs during lentiviral gene delivery.

Multilineage polyclonal engraftment of Cal-1 gene-modified cells and in vivo selection after SHIV infection in a nonhuman primate model of AIDS.

We have focused on gene therapy approaches to induce functional cure/remission of HIV-1 infection. Here, we evaluated the safety and efficacy of the clinical grade anti-HIV lentiviral vector, Cal-1, in pigtailed macaques (Macaca nemestrina). Cal-1 animals exhibit robust levels of gene marking in myeloid and lymphoid lineages without measurable adverse events, suggesting that Cal-1 transduction and autologous transplantation of hematopoietic stem cells are safe, and lead to long-term, multilineage engraftment following myeloablative conditioning.

Controlling HIV-1: Non-Coding RNA Gene Therapy Approaches to a Functional Cure.

The current treatment strategy for HIV-1 involves prolonged and intensive combined antiretroviral therapy (cART), which successfully suppresses plasma viremia. It has transformed HIV-1 infection into a chronic disease. However, despite the success of cART, a latent form of HIV-1 infection persists as integrated provirus in resting memory CD4(+) T cells.

Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.

We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis.

CCR5 as a natural and modulated target for inhibition of HIV.

Human immunodeficiency virus type 1 (HIV-1) infection of target cells requires CD4 and a co-receptor, predominantly the chemokine receptor CCR5. CCR5-delta32 homozygosity results in a truncated protein providing natural protection against HIV infection-this without detrimental effects to the host-and transplantation of CCR5-delta32 stem cells in a patient with HIV ("Berlin patient") achieved viral eradication. As a more feasible approach gene-modification strategies are being developed to engineer cellular resistance to HIV using autologous cells.

Preclinical safety and efficacy of an anti-HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor.

Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA) for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46.

Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains.

Phase I/II Clinical Trials Using Gene-Modified Adult Hematopoietic Stem Cells for HIV: Lessons Learnt.

Gene therapy for individuals infected with HIV has the potential to provide a once-only treatment that will act to reduce viral load, preserve the immune system, and mitigate cumulative toxicities associated with highly active antiretroviral therapy (HAART). The authors have been involved in two clinical trials (phase I and phase II) using gene-modified adult hematopoietic stem cells (HSCs), and these are discussed as prototypic trials within the general field of HSC gene therapy trials for HIV. Taken as a group these trials have shown (i) the safety of both the procedure and the anti-HIV agents themselves and (ii) the feasibility of the approach.

The use of cell-delivered gene therapy for the treatment of HIV/AIDS.

HIV/AIDS is a disease that impairs immune function, primarily by decreasing T-lymphocyte count. Its progression can be contained by highly active antiretroviral therapy (HAART), but there are side effects that can be severe, and the development of resistance often forces the physician to modify the HAART regimen. There are no vaccines available for HIV.

Mathematical modelling of the impact of haematopoietic stem cell-delivered gene therapy for HIV.

Background: Gene therapy represents a new treatment paradigm for HIV that is potentially delivered by a safe, once-only therapeutic intervention.

Methods: Using mathematical modelling, we assessed the possible impact of autologous haematopoietic stem cell (HSC) delivered, anti-HIV gene therapy. The therapy comprises a ribozyme construct (OZ1) directed to a conserved region of HIV-1 delivered by transduced HSC (OZ1+HSC).

Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells.

Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1-infected adults who received a tat-vpr-specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events.

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients.

Background: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2.

Methods: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors.

Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and lymphoid reconstitution in human immunodeficiency virus type-1-infected patients.

A phase I gene transfer clinical study was undertaken to examine the ability to introduce a potential anti-human immunodeficiency virus (HIV) gene therapeutic into hematopoietic progenitor cells (HPC), thereby contributing to multilineage engraftment. The potential therapeutic effect of genetically modifying HPC with protective genes in HIV-infected adults depends in part on the presence of adult thymic activity and myeloid capacity in the setting of HIV replication. Herein we report the presence and expression of a retroviral vector encoding an anti-HIV-1 ribozyme in mature hematopoietic cells of different lineages, and de novo T-lymphocyte development ensuing from genetically engineered CD34(+) HPC.

Critical steps in the implementation of hematopoietic progenitor-cell gene therapy using ribozyme vectors: a laboratory protocol.

The implementation of a hematopoietic progenitor-cell gene-therapy program involves the performance of laboratory procedures and compliance with the current code of Good Manufacturing Practices. This chapter explains the multiple laboratory steps used in our recent Phase I gene transfer study for HIV. This study employed a retroviral vector to deliver an anti-HIV ribozyme to CD34+ hematopoietic progenitor cells.

Clinical gene therapy research utilizing ribozymes: application to the treatment of HIV/AIDS.

Antiretroviral drug therapy can effectively reduce the viral load, and is associated with a degree of immune reconstitution in human immunodeficiency virus (HIV)-infected patients. However, the presence of a latent viral reservoir, the development of drug resistance, drug toxicity, and compliance problems are obstacles that impede full eradication of HIV through drug therapy. The cellular introduction of genetic elements that are capable of inhibiting HIV replication is conceptually appealing as a potential new treatment paradigm for acquired immunodeficiency syndrome (AIDS).