The Pharmaceutical Journal
Vol 270 No 7241 p412-413
22 March 2003

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Joint Pharmaceutical Analysis Group

Regulatory status and analytical challenges of gene therapy

Professor David Kerr, department of clinical pharmacology, University of Oxford, gave an overview of the rationale and efficacy of gene therapy with particular reference to his own involvement in gene therapy for liver cancer. There were several approaches to cancer treatment using gene therapy, including replacement of a damaged gene, immunotherapy and selective killing. More specifically, the latter approach could be typified by the viral delivery of enzymes for drug therapy. The enzyme released in the tumour converts a non-toxic prodrug to the toxic entity resulting in local cell death. Thus, CB1954 is converted by expressed nitroimidazole reductase to a toxic bifunctional alkylating agent. Routes to tumour selectivity exist on three levels: regional delivery, such as via the local blood supply or intratumourally; molecular targeting of vectors; and tumour specific expression of the therapeutic gene.

Professor Kerr has developed the use of adenoviral nitroimidazole reductase for use in gene therapy of cancer. Five years of preclinical activity has been expended in demonstrating in vitro sensitisation, cures in hepatoma xenografts, survival prolongation in peritoneal carcinomatosis and minimisation of the bystander effect.

Any gene therapy programme is subject to regulatory procedures through the Gene Therapy Advisory Committee, Medicines Control Agency, Health and Safety Executive, and local research ethics committees. For the trials, patients with hepatic cancer amenable to surgery, with reasonable haematological and biochemical function and no active adenoviral infection were eligible. The endpoints for monitoring the therapy included safety and toxicity, expression of the enzyme, and distribution of the virus. The safety and toxicity issue included both the virus and the prodrug. In a group of 12 patients given escalating doses of the virus until enzyme expression there was no hepatic dysfunction, myelosuppression or systemic toxicity. The enzyme was expressed with increasing dose of the virus. A phase I study of CB1954 resulted in a recommended dose of 24mg/m3 of this prodrug to achieve active but tolerable systemic blood levels. Thus with this well-tolerated virus and acceptable enzyme expression (up to 25 per cent), the next step is to apply the virus and prodrug in advanced disease.

Systemic delivery

Gene therapy is powerful, controllable and potentially curative for genetic disease said Dr Leonard Seymour, of the Radcliffe Infirmary, University of Oxford, but there remains the problem of how to deliver the therapy. Nearly all cancer gene therapy clinical trials involve local administration. Adenovirus, the quintessential vector for gene delivery, has the advantages of being powerful (potentially one virus per cell activity), being active in non-dividing cells and having a high transgene capacity. However the disadvantages include broad cellular tropism, unsuitability for systemic use due to rapid clearance, and a susceptibility to antibody neutralisation.

Strategies for modifying viral gene delivery vectors include genetic modification (peptide incorporation, C-terminal modification), the use of bi-specific molecules (bi-specific antibody, antibody-ligand conjugate), non-covalent polymer modification (polyethylene glycol coating, micro-encapsulation) and covalent polymer modification (polyethyleneglycolation).

Liver has been shown to be the main site of virus uptake in naive mice and ligation of hepatic blood flow can be used to extend the circulation and hence the utility of systemic delivery of adenovirus.

Dr Seymour concluded that, although the promise of adenovirus for cancer gene therapy is limited by problems with its initial delivery, polymer-coating provides a versatile way to endow new tropisms and protection from neutralising antibodies. Coated viruses can be engineered to infect via target cell-associated receptors and systemic delivery to disseminated targets looks increasingly feasible. An important additional consideration is that a modified phenotype is not heritable.

Quality and stability

The overall aim of quality control, good manufacturing practice and quality assurance is to protect the patient said Roy Cowell (Cobra Biomanufacturing). Pharmaceutical analysts are adept in characterising and controlling new chemical entities where routes and mechanisms of synthesis are well-characterised and understood and the final product is likely to be obtained in at least 70 per cent yield. In contrast, manufacturing procedures for gene therapy products are complex and highly dependent on the relatively unpredictable behaviour of cell lines. In general the final product, be it plasmid DNA or viral particles, will be less than 1 per cent of the biomass generated during manufacture, possibly with much larger quantities of protein, RNA, host chromosomal DNA and lipopolysaccharides being present. Product yield and quality are heavily dependent on providing the optimum conditions consistently.

Understanding the challenge to downstream purification strategies highlights the need for appropriate quality control, not only in testing of the bulk active substance but also in the manufacturing process itself. Process-related impurities are often present in the same quantities as the desired product. This presents a challenge for the analytical methodology applied in monitoring levels of residuals. Low yield of product necessitates high biomass production, making quality control procedures important for effective scale up and optimisation of column-based purification procedures. During manufacturing, equipment is likely to be exposed to lysed cells and, where the equipment is not disposable, the effectiveness of cleaning procedures should be demonstrated.

Gene therapy procedures are diverse, ranging from combination therapies to vaccinations. In assessment of the safety of gene therapy products guidance is derived from the regulatory authorities, advisory committees and the pharmacopoeias. In terms of assessing intrinsic (and extrinsic) quality the analytical toolbox is derived from knowledge of the properties of the product and its related substances. The mechanism of action or functionality of gene therapy products is often modelled through in vivo and in vitro techniques, although these tests are not a measure of efficacy. Stability testing, and understanding the routes of degradation may dictate how a product may be presented to the clinician and used in the patient. Stability studies should address the identified key aspects of the product that impact on the dose and therefore patient safety.

FDA perspectives

The presentation by Dr Steve Bauer, of the Food and Drug Administration in the United States, was given via a video link from his office, an innovation that worked remarkably smoothly. Dr Bauer emphasised that the FDA operated an informal system whereby researchers and clinicians contemplating gene therapy could hold preliminary discussions with FDA officials even before an Investigational New Drug Application was filed.

For the analyst, the most important feature of Dr Bauer's presentation was the evaluation of biodistribution or expression studies in animals and in patients. The most common approach was to use the polymerase chain reaction (PCR) to detect the vector genome, or reverse transcriptase PCR to detect the product. Biodistribution studies are designed to address the potential for germline alteration, and the potential for toxicity in other tissues and organs. The polymerase chain reaction provides high sensitivity (a single copy is the theoretical limit of detection), and only a small amount of material is required. However, false positives and false negatives are common at low levels.

Dr Bauer contended that PCR was deceptively easy with several common pitfalls: sensitivity or detection limits need to be established. Contamination, either during tissue harvest, or from carry-over is also a hazard. An understanding of the efficiency of the PCR and the understanding its reaction kinetics cannot be underestimated. The sample itself, for example, liver or gonad tissue, will affect the reaction kinetics and lead to inaccurate results unless a proper standard curve is constructed.

The criteria for evaluating PCR used as an analytical method are the same as for any other classical method: specificity, by demonstration of detection of the desired target (expected size, expected sequence, unique target when possible); limit of detection by vector dilution and sufficient sampling at low copy number; and sensitivity by studies with added analyte.

Minimal PCR recommendations for biodistribution studies were to analyse three samples per tissue, containing 1mg genomic DNA in each, two samples run without any added control and one sample run with added control; sensitivity should be better than 100 copies of vector per mg genomic DNA, although this figure may be different for different situations.

The Gene Therapy Advisory Committee

"Reports of my death have been greatly exaggerated!" Mark Twain was talking about himself, but Dr Jayne Spink, genetic science policy, Department of Health, said the comment could equally apply to gene ther-apy; despite gloomy reports in the press and television, gene therapy was healthy and vibrant. Dr Spink explained that the Gene Therapy Advisory Committee (GTAC) had come into being following the 1992 Clothier report, which recommended the establishment of such a national supervisory body. The committee is complementary to other UK regulatory authorities.

Members are appointed by ministers and the committee acts both as an ethical committee and as a ministerial advisory board, providing for national oversight, advice to researchers and the organisation of workshops, conferences and working parties.

One of GTAC's main responsibilities is to advise on the ethical acceptability and scientific or medical merit of any proposals. The main body of a submission to GTAC should include the nature of material to be administered, reference to prior studies, a safety-benefit assessment, the clinical protocol, site and investigator details, proposed follow-up procedures, and informed consent documentation. The study must be scientifically justified and validated with preclinical data, the natural history of the disease, other available treatments, informed patient consent and potential efficacy and safety.

However, gene therapy is a rapidly evolving field, which means that the control must be flexible, there must be mechanisms to address new advances and the guidelines must be living documents. GTAC must keep up with events. For example, a proposal to use gene therapy in the womb would need to be critically assessed and GTAC concluded that it would consider such procedures provided there was a clear advantage over postnatal therapy and that there was a clear advantage over therapy with unmodified cells. However, direct gene therapy would be off limits when there were scientific and ethical concerns, or there was a possibility of non-target effects.

Contained use regulations

A number of European directives relate to gene therapy. Dr Patrick Seechurn, Health and Safety Executive, concentrated in his talk on the Genetically Modified Organisms (GMO) (Contained Use) Regulations and their operation in a clinical setting.

The regulations require that contact with GMO is limited through the use of biological, chemical and physical barriers, and that risk to human health and the environment is considered in the risk assessment.

"Contained use" is defined as any activity in which genetically modified organisms are cultured, stored, transported, destroyed, disposed of or used in any other way and for which physical, chemical or biological barriers are used to limit contact with humans or the environment to ensure a high degree of safety. Thus the regulations apply to personnel involved in gene therapy although not, paradoxically, to the patient receiving the therapy.

The key requirements of the regulations include the establishment of a genetic modification safety committee, risk assessment via a classification system, reduction of worker and environmental exposure to the lowest level reasonably practicable, and notification.

It is a requirement of the regulations that the activity is classified into one of four classification groups based on the containment measures that are needed to control or manage the risks associated with the activity. However, the regulations do not specify containment measures for clinical trials. The classification of the activity based on the laboratory risk assessment is a starting point for classifying the gene therapy trial.

For highly disabled gene therapy vectors with a narrow host range expressing a non-harmful insert, the physical containment measures are likely to be minimal and consequently classified as a class 1 activity that would equate to an activity of negligible risk. Where replication-competent vectors are used or vectors which produce significant shedding and expression of a potent biological payload, then additional containment measures are likely to be required to limit exposure. The Health and Safety Executive is in the process of developing guidance to assist users of genetically modified organisms in classifying gene therapy trials and on meeting the requirements of the regulations.

Gene therapy products in hospitals

What happens when we finally have a gene therapy product that works in the patient? Richard Bateman, Guy' and St Thomas' NHS Trust, London, asked. The answer is that we will have to use it in a hospital setting and it is useful to look at the current situation and what needs to be done in practical terms. The current concerns are for patient and operator protection, protection of the product and protection of other products and the environment.

The risks associated with gene therapy vary among individual products with a wide range of actions. Knowledge therefore is required on the individual products being handled, and practical and realistic controls are needed on a case-by-case basis. General considerations include simple reconstitution and dilution of the product, complex manipulation and processing, whether to use open or closed systems, segregation of vector production, biological safety levels for vector production and design of vector production facilities. Good technique and appropriate equipment must be used to minimise exposure to agents, and separation from other production activities (cabinets, rooms, air handling) may be required. Sometimes new requirements may be in conflict with existing requirements. For example, aseptic laboratories are operated under positive pressure to keep out contamination, whereas recommendations for gene therapy facilities require negative pressure.

In the absence of purpose-built laboratories and hospitals, most facilities will have to adapt their current buildings and equipment. However it must be acknowledged that sometimes it will not be possible to handle certain products, in which case capital investment will be required.

In the future gene therapy will develop in its nature, in the scale and number of products and will involve other new technologies. There will be new requirements for pharmacy services (both for gene therapy and conventional medicines) and these demands will have to be met by the planning of new facilities.