Cell and gene therapy manufacturing capabilities in Australia and New Zealand.

Cell and gene therapy products are rapidly being integrated into mainstream medicine. Developing global capability will facilitate broad access to these novel therapeutics. An initial step toward achieving this goal is to understand cell and gene therapy manufacturing capability in each region. We conducted an academic survey in 2018 to assess cell and gene therapy manufacturing capacity in Australia and New Zealand. We examined the following: the number and types of cell therapy manufacturing facilities; the number of projects, parallel processes and clinical trials; the types of products; and the manufacturing and quality staffing levels. It was found that Australia and New Zealand provide diverse facilities for cell therapy manufacturing, infrastructure and capability. Further investment and development will enable both countries to make important decisions to meet the growing need for cell and gene therapy and regenerative medicine in the region.

Enhancers are major targets for murine leukemia virus vector integration.

UNLABELLED: Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34(+) hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE: We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors.

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.

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). OZ1+HSC contributes to the CD4+ T lymphocyte and monocyte/macrophage cell pools that preferentially expand under the selective pressure of HIV infection. The model was used to predict the efficacy of OZ1 in a highly active antiretroviral therapy (HAART) naïve individual and a HAART-experienced individual undergoing two structured treatment operations. In the standard scenario, OZ1+HSC was taken as 20% of total body HSC. RESULTS: For a HAART-naïve individual, modelling predicts a reduction of HIV RNA at 1 and 2 years post-OZ1 therapy of 0.5 log(10) and 1 log(10), respectively. Eight years after OZ1 therapy, the CD4+ T-lymphocyte count was 271 cells/mm(3) compared to 96 cells/mm(3) for an untreated individual. In a HAART-experienced individual HIV RNA was reduced by 0.34 log(10) and 0.86 log(10) at 1 and 2 years. The OZ1 effect was maximal when both CD4+ T lymphocytes and monocytes/macrophages were protected from successful, productive infection by OZ1. CONCLUSIONS: The modelling indicates a single infusion of HSC cell-delivered gene therapy can impact on HIV viral load and CD4 T-lymphocyte count. Given that gene therapy avoids the complications associated with HAART, there is significant potential for this approach in the treatment of HIV.

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.

Co-Culture of Human Bone Marrow Cells with a Mastocytosis Cell Strain Induces Mast Cell/Basophil Differentiation.

Metachromatic staining cells with mast cell and basophil characteristics were grown from normal human bone marrow, both in co-culture with a human mast cell-like strain (HBM-M) and when maintained in the presence of conditioned medium derived from this cell strain (HBM-M-CM). In co-culture with HBM-M, >25% of the bone marrow cells stained metachromatically with toluidine blue from day 20-40 compared to <10% in control cultures. Half the cells were stained by the basophil-specific antibody Bsp-1 and 50% bound IgE. In the presence of 50% HBM-M-CM, up to 60% of marrow-derived cells were positive for toluidine blue, while 15-36% of cells bound Bsp-1. Conditioned medium from HBM-M cells did not contain IL-3, IL-5, or GM-CSF but did contain approximately 0.1 ng/ml stem cell factor. HBM-M cells appear to secrete an unidentified growth factor(s) capable of promoting the production and/or differentiation from normal human bone marrow of metachromatic staining cells in liquid culture.

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). 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.

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.

Raising the standard: changes to the Australian Code of Good Manufacturing Practice (cGMP) for human blood and blood components, human tissues and human cellular therapy products.

In Australia, manufacture of blood, tissues and biologicals must comply with the federal laws and meet the requirements of the Therapeutic Goods Administration (TGA) Manufacturing Principles as outlined in the current Code of Good Manufacturing Practice (cGMP). The Therapeutic Goods Order (TGO) No. 88 was announced concurrently with the new cGMP, as a new standard for therapeutic goods. This order constitutes a minimum standard for human blood, tissues and cellular therapeutic goods aimed at minimising the risk of infectious disease transmission. The order sets out specific requirements relating to donor selection, donor testing and minimisation of infectious disease transmission from collection and manufacture of these products. The Therapeutic Goods Manufacturing Principles Determination No. 1 of 2013 references the human blood and blood components, human tissues and human cellular therapy products 2013 (2013 cGMP). The name change for the 2013 cGMP has allowed a broadening of the scope of products to include human cellular therapy products. It is difficult to directly compare versions of the code as deletion of some clauses has not changed the requirements to be met, as they are found elsewhere amongst the various guidelines provided. Many sections that were specific for blood and blood components are now less prescriptive and apply to a wider range of cellular therapies, but the general overall intent remains the same. Use of 'should' throughout the document instead of 'must' allows flexibility for alternative processes, but these systems will still require justification by relevant logical argument and validation data to be acceptable to TGA. The cGMP has seemingly evolved so that specific issues identified at audit over the last decade have now been formalised in the new version. There is a notable risk management approach applied to most areas that refer to process justification and decision making. These requirements commenced on 31 May 2013 and a 12 month transition period applies for implementation by manufacturers. The cGMP and TGO update follows the implementation of the TGA regulatory biologicals framework for cell and tissue based therapies announced in 2011. One implication for licenced TGA facilities is that they must implement the 2013 cGMP, TGO 88 and other relevant TGOs together, as they are intricately linked. This review is intended to assist manufacturers by comparing the 2000 version of the cGMP, to the new 2013 cGMP, noting that the new Code extends to include human cellular therapy products.

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

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.

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.

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.

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. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.