Somatic Cell Gene Therapy Research Articles

Draft of FDA's Points to Consider in Human Somatic Cell Therapy and Gene Therapy (1991)

Draft of FDA's Points to Consider in Human Somatic Cell Therapy and Gene Therapy (1991)

Regulatory Concerns in Human Gene Therapy

Gene therapy in humans is now being undertaken in an investigational setting. Such therapy involves the administration of biological products to human patients. A document entitled, "Points to Consider in Human Somatic Cell Therapy and Gene Therapy" has been prepared by the Center for Biologics Evaluation and Research (CBER) of the Food and Drug Administration (FDA) and is published elsewhere in this issue. This paper provides explanatory material about the CBER regulatory process and the scientific and regulatory basis for the requests for data in that document.

Human Gene Therapy: Ethics and Public Policy

The first three human gene transfer/therapy clinical protocols are now underway after having been subjected to an extensive review process by the Recombinant DNA Advisory Committee (RAC) and its Human Gene Therapy Subcommittee. The "Points to Consider" document developed by the RAC established the framework for evaluating genetic intervention protocols. This review process is taking place in a broader social context. Public attitude surveys in this country have indicated a general lack of knowledge in the area of genetic engineering but an acceptance of somatic-cell gene therapy as treatment for disease. Internationally, numerous policy statements on human genetic intervention have been published, all of which support the moral legitimacy of somatic-cell gene therapy for the cure of disease. The debate over the ethical issues related to somatic-cell gene therapy has evolved over a ten-year-period. The time has now come to begin a formal public process for the ethical assessment of germ-line genetic intervention.

Human gene therapy

Somatic cell gene therapy for the correction of many human genetic diseases is now technically possible. We review several methods of gene transfer that have been successfully used in animal studies, and discuss the promise and potential limitations of these methods in the treatment of human genetic diseases.

An in Vivo Model of Somatic Cell Gene Therapy for Human Severe Combined Immunodeficiency

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency (SCID), a candidate genetic disorder for somatic cell gene therapy. Peripheral blood lymphocytes from patients affected by ADA- SCID were transduced with a retroviral vector for human ADA and injected into immunodeficient mice. Long-term survival of vector-transduced human cells was demonstrated in recipient animals. Expression of vector-derived ADA restored immune functions, as indicated by the presence in reconstituted animals of human immunoglobulin and antigen-specific T cells. Retroviral vector gene transfer, therefore, is necessary and sufficient for development of specific immune functions in vivo and has therapeutic potential to correct this lethal immunodeficiency.

Stable antiviral expression in BALB/c 3T3 cells carrying a beta interferon sequence behind a major histocompatibility complex promoter fragment

We are exploring the use of interferon (IFN) genes for somatic cell gene therapy and have investigated the possibility of transforming cells into low constitutive IFN producers over prolonged periods of time without impeding cell survival or replication. Here we report the possibility of conferring a permanent antiviral state to cells by introducing an IFN-beta gene with a modified transcriptional control. This was achieved by placing the murine IFN-beta-coding sequence behind the IFN-inducible 0.6-kb XhoII-NruI promoter region of the H-2Kb major histocompatibility complex gene. BALB/c 3T3 cells that are normally permissive for virus infection were transformed with this construction, and 14 of the 21 clonal cell lines obtained displayed different levels of enhanced resistance to the replication of vesicular stomatitis virus, encephalomyocarditis virus, and Semliki Forest virus. The permanent antiviral state was dependent on the presence of new IFN-beta fragments in Southern blots and was accompanied by very low constitutive synthesis of IFN-beta, and the presence of construct-derived IFN mRNA could be demonstrated only after polymerase chain reaction amplification of cDNA. The antiviral state was stable over a 9-month period in culture, corresponding to about 250 cell generations, and did not affect cell replication, since the average population doubling time of these cells was not significantly different from that of the control clone. Twenty-nine control clonal cell lines, stably transformed with either the neo gene alone (22 lines) or the neo gene together with a mutated murine IFN-beta gene coding for an inactive protein (7 lines), did not develop stable antiviral expression. We conclude that low constitutive synthesis of autocrine IFN-beta is sufficient to induce a permanent antiviral state, is compatible with cell survival and replication, and therefore merits further exploration for use in somatic cell gene therapy.

Gene Therapy: Where to Draw the Line

The four classifications of genetic manipulation (somatic cell gene therapy, germ-line gene therapy, enhancement genetic engineering, eugenic genetic engineering) are reviewed from an ethical viewpoint. Immediately, it needs to be recognized that words like "therapy," "enhancement," and "eugenic" already possess an emotional framework, so that careful reasoning is made more difficult. In each of the areas of genetic manipulation, it can be argued that circumstances could be imagined that would either justify or not justify proceeding. Based on our present state of knowledge, the line should be drawn at not carrying out gene therapy of any type except in specific cases that are carefully evaluated in advance. With increased knowledge, the line should be moved to embrace appropriate situations.

Evolution of Ethical Debate about Human Gene Therapy

Ethical issues generally evolve through four stages: threshold, open conflict, extended debate, and adaptation. The history of the ethical debate on human gene therapy is examined. The threshold was the Nirenberg appeal in 1967. The open conflict centered around two early controversial cases: those of Rogers and Cline. The extended debate has lasted from 1980 to the present, but now adaptation, i.e., a public policy, for somatic cell gene therapy is emerging.

Transkaryotic implantation and the somatic cell gene therapy of diabetes

Transkaryotic implantation and the somatic cell gene therapy of diabetes

Genetics and Human Malleability

Genetics and Human Malleability Just how much can, and should we change human nature ... by genetic engineering? Our response to that hinges on the answers to three further questions: (1) can we do now? Or more precisely, what are we doing now in the area of human genetic engineering? (2) will we be able to do? In other words, what technical advances are we likely to achieve over the next five to ten years? (3) should we do? I will argue that a line can be drawn and should be drawn to use gene transfer only for the treatment of serious disease, and not for any other purpose. Gene transfer should never be undertaken in an attempt to enhance or improve human beings. Can We Do? In 1980 John Fletcher and I published a paper in the New England Journal of Medicine in which we delineated what would be necessary before it would be ethical to carry out human gene therapy. [1] As with any other new therapeutic procedure, the fundamental principle is that it should be determined in advance that the probable benefits outweigh the probable risks. We analyzed the risk/benefit determination for somatic cell gene therapy and proposed three questions that need to have been answered from prior animal experimentation: Can the new gene be inserted stably into the correct target cells? Will the new gene be expressed (that is, function) in the cells at an appropriate level? Will the new gene harm the cell or the animal? These criteria are very similar to those required before use of any new therapeutic procedure, surgical operation, or drug. They simply require that the new treatment should get to the area of disease, correct it, and do more good than harm. A great deal of scientific progress has occurred in the nine years since that paper was published. The technology does now exist for inserting genes into some types of target cells. [2] The procedure being used is called retroviral-mediated gene transfer. In brief, a disabled murine retrovirus serves as a delivery vehicle for transporting a gene into a population of cells that have been removed from a patient. The gene-engineered cells are then returned to the patient. The first clinical application of this procedure was approved by the National Institutes of Health and the Food and Drug Administration on January 19, 1989. [3] Our protocol received the most thorough prior review of any clinical protocol in history: It was approved only after being reviewed fifteen times by seven different regulatory bodies. In the end it received unanimous approval from every one of those committees. But the simple fact that the NIH and FDA, as well as the public, felt that the protocol needed such extensive review demonstrates that the concept of gene therapy raises serious concerns. We can answer our initial question, What can we do now in the area of human genetic engineering?, by examining this approved clinical protocol. Gene transfer is used to mark cancer-fighting cells in the body as a way of better understanding a new form of cancer therapy. The cancer-fighting cells are called TIL (tumor-infiltrating-lymphocytes), and are isolated from a patient's own tumor, grown up to a large number, and then given back to the patient along with one of the body's immune growth factors, a molecule called interleukin 2 (IL-2). The procedure, developed by Steven Rosenberg of the NIH, is known to help about half the patients treated. [4] The difficulty is that there is at present no way to study the TIL once they are returned to the patient to determine why they work when they do work (that is, kill cancer cells), and why they do not work when they do not work. The goal of the gene transfer protocol was to put a label on the infused TIL, that is, to mark these cells so that they could be studied in blood and tumor specimens from the patient over time. The TIL were marked with a vector (called N2) containing a bacterial gene that could be easily identified through recombinant DNA techniques. …

Human gene therapy: why draw a line?

Despite widespread agreement that it would be ethical to use somatic cell gene therapy to correct serious diseases, there is still uneasiness on the part of the public about this procedure. The basis for this concern lies less with the procedure's clinical risks than with fear that genetic engineering could lead to changes in human nature. Legitimate concerns about the potential for misuse of gene transfer technology justify drawing a moral line that includes corrective germline therapy but excludes enhancement interventions in both somatic and germline contexts.

Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector

Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene ( tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457–460]. Infection of HPRT - cultured rat neuronal cells with these vectors resulted m transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.

A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells.

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.

A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells.

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.

Genetic correction of hereditary disease.

Several hereditary disorders may be potentially correctable by the introduction and incorporation of the normal gene into human tissues using a variety of systems. Although technical issues surrounding integration, stable expression and potential insertional mutagenesis to the treated cells has not yet been fully resolved, enough scientific progress has already been made to consider somatic cell gene therapy acceptable from both the scientific and ethical viewpoints. Tissue-specific stem cell and embryonic stem cell transplantation will allow therapy earlier in the developing embryo. As technical problems are eliminated, these procedures will become morally permissible, as they will allow the correction of devastating hereditary disease.

Technology assessment of a gene therapy

One suggested motive for massive international research programmes (financially comparable to the moon landings) into the analysis of the human genome is the potential for improved medical diagnosis and treatment of the 4,000 known singlegene hereditary disorders. This paper presents a technology assessment framework containing some costs and benefits to analyse the merits of attempting to apply any new knowledge from this programme to the development of a novel medical treatment: the use of what is called somatic cell gene therapy (an application of genetic engineering) to a most unpleasant condition affecting some one in 16,000 live birth's (Lesch-Nyhan syndrome). The authors' conclusion is that any such treatment seems unlikely to work well, for technical reasons; that the consequential benefits to sufferers are unlikely to be great. Financial and non-financial costs and risks will arise for both patients (from side-effects, etc) and for the wider community. Thus this aspect of the human genome programme seems unlikely to increase social welfare.

Human Gene Therapy

This book is based on a meeting and symposium by acknowledged eminent genetic experts who were convened by the Councils of the National Academy of Sciences and the Institute of Medicine in October 1986. purpose was to delineate the level of the science of gene therapy, additional technical difficulties to be mastered, and the public policy process needed prior to any efforts to apply gene therapy. symposium title was Human Somatic Cell Gene Therapy: Prospects for Treating Inherited Diseases. This book summarizes the presentations at the meeting and subsequent workshop, and the author provides acknowledgment of the contributions of each of the principal speakers at the conclusion of each chapter. This small volume is extremely well written, and the reader should not be deterred by the minimal, initial melodramatic approach, which might have been designed to capture the attention of nonprofessional persons, eg, The baby's caesarean birth—on December

The Rome Bioethics Summit

Scientists at the Fifth International Summit Conference on Bioethics, hosted by the Italian government in April 1988, were overwhelmingly supportive of efforts now underway to map and sequence the human genome because of its great benefit in preventing and treating disease. The meeting also concluded that somatic cell gene therapy should be classified with other medical experimentation and that medical indications or ethical justifications do not now exist for intentional manipulation of human germline cells.

An alternative approach to somatic cell gene therapy.

Mouse primary skin fibroblasts were infected with a recombinant retrovirus containing human factor IX cDNA. Bulk infected cells capable of synthesizing and secreting biologically active human factor IX protein were embedded in collagen, and the implant was grafted under the epidermis. Sera from the transplanted mice contain human factor IX protein for at least 10-12 days. Loss of immunoreactive human factor IX protein in the mouse serum is not due to graft rejection. Instead, the mouse serum contains anti-human factor IX antibodies, which react with the protein. We suggest that retroviral-infected primary skin fibroblasts offer an alternative approach to somatic cell gene therapy.

Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells.

Recombinant retroviral genomes encoding a chromosomal human beta-globin gene have been used to transduce murine haematopoietic stem cells in vitro. After permanent engraftment of lethally irradiated recipients with the transduced cells, the human beta-globin gene is expressed at significant levels only within the erythroid lineage. These results indicate that it is possible to obtain stable expression of exogenous chromosomal DNA sequences introduced into mature haematopoietic cells in vivo via stem cell infection, and that human disorders of haemoglobin production may be more feasible candidates for somatic cell gene therapy than previously suspected.