Janet L. Macpherson

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. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product.

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. Sustained output of vector-containing mature myeloid and T-lymphoid cells was detected even in patients with multidrug-resistant infection. In addition, the study showed that the degree of persistence of gene-containing cells was dependent on transduced HPC dose. These novel findings support the concept of gene therapy as a modality to effect immune reconstitution with cells engineered to inhibit HIV replication and this report represents the first demonstration of long-term maintenance of a potential therapeutic transgene in HIV disease.

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

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.

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. They point to the requirement for (i) the ability to transduce and infuse as many as possible gene-containing HSC and/or (ii) high engraftment and in vivo expansion of these cells, (iii) potentially increased efficacy of the anti-HIV agent(s) and (iv) automation of the cell processing procedure.

Clinical Gene Therapy Research Utilizing Ribozymes

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). In theory, this approach can lead to the development of regenerated hematopoiesis with cells that inhibit viral replication and are protected from the pathogenic effects of HIV. Ribozymes are catalytic RNA molecules that can efficiently and selectively cleave target RNA. By ex vivo retroviral transduction, we have introduced a HIV-1 tat gene-targeted ribozyme (RRz2) and a control construct (LNL6) into granulocyte-colony-stimulating factor (G-CSF) mobilized CD34+ hematopoietic progenitor cells (HPC). Transduced autologous CD34+ cells (an approximately equal mix of RRz2 and LNL6) were infused in 10 patients in this Phase I study. After a median follow-up of 2.5 yr, gene presence and expression were detected by a sensitive polymerase chain reaction (PCR) assay in a transduced-CD34+ cell dose-dependent manner. In this chapter, we describe general considerations related to HIV hematopoietic progenitor-cell gene therapy trial design, implementation, and safety, with an emphasis on the critical steps of this process, namely vector production and characterization, target-cell selection, transduction, final product release testing, and evaluation of vector presence.

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

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

T-lymphocyte perturbation following large-scale apheresis and hematopoietic stem cell transplantation in HIV-infected individuals

Analysis and mathematical modeling of T-lymphocyte perturbation following administration of granulocyte colony stimulating factor (G-CSF) and two large-scale aphereses are reported. 74 HIV-1 positive antiretroviral-treated individuals were infused with gene- or sham-transduced CD34+ hematopoietic stem cells (HSC) in a Phase II clinical trial. T cell numbers were examined in four phases: 1) during steady state; 2) increases in peripheral blood (PB) following G-CSF administration; 3) depletion post-aphereses and 4) reconstitution post HSC infusion. The present analysis provides the first direct estimate of CD4+ T cell distribution and trafficking in HIV-infected individuals on stable HAART, indicating that CD4+ T lymphocytes in PB represent 5.5% of the pool of CD4+ T lymphocytes that traffic to PB.

Immortalization and characterization of human cell lines with mast cell and monocytic properties

Summary. We have previously derived a cell strain which had both mast cell and monocytic properties from the bone marrow of a child with diffuse cutaneous mastocytosis. This cell strain, termed HBM‐M, consisted of two cell populations both of which possessed certain ultrastructural, cytochemi‐cal and surface phenotypic features of degranulated mast cells. The cells also displayed cytochemical and surface phenotypic features of monocytes. These cells may represent a common bone marrow derived mast cell/monocyte precursor. Studies of human mast cells have been hindered by the fact that it is difficult to establish such cells in long‐term culture. Thus, we sought to immortalize HBM‐M cells by introducing Simian virus 40 large T‐antigen. Following transfection by the strontium phosphate technique, transformed cells were selected, expanded and passaged until the cells entered a non‐proliferative phase termed crisis. Certain clones passed through crisis 3 months later and by this means two immortal cell lines, HBM‐MI‐1 and HBM‐MI‐2, were obtained. The criterion for immortality was growth for greater than 100 population doublings. The immortal cell lines retained some, but not all. of the mast cell and monocytic properties of the original HBM‐M cell strain. The immortalization of the cell strain HBM‐M provides an opportunity to investigate the mast cell and monocytic properties of these cells, and the apparent relationship between mast cells and monocytes.

Clinical potential of gene therapy: towards meeting the demand

Since the discovery that new genetic material could be transferred into human cells resulting in induced expression of genes and proteins, clinicians and scientists have been working to harness the technology for clinical outcomes. This article provides a summary of the current status of developments within the broad discipline of clinical gene therapy. In pursuing the treatment of diverse clinical conditions, a wide variety of therapeutics, each tailor‐made, may be required. Gene therapy offers the possibility of accurately and specifically targeting particular genetic abnormalities through gene correction, addition or replacement. It represents a compelling idea that adds a new dimension to our portfolio of credible therapeutic choices.

Cellular therapy in the Asia-Pacific region. A guide for the future pathologist.

Summary The Asia-Pacific region includes a large number of countries offering a broad range and quality of healthcare services. Almost every country in the region offers at least some cellular therapies, from the highly regulated countries like Japan, Korea and Australia, through to countries where the oversight is less formal. The key healthcare drivers for this sector are the ageing population, obesity epidemic, organ donation statistics and the emergence of personalised medicine. This is a rapidly advancing field with breakthroughs announced regularly. The Asia-Pacific region is poised to become a world leader in the provision of this new generation of therapeutic options in a safe and standardised manner.