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Gene therapy research paper

Thus, the standard mouse model is inadequate for the quantitative assessment of oncogenic risk. Since all medical interventions carry some risk, it is incumbent on gene therapy researchers to establish quantitative measures of this risk. For example, it may be found for a certain disease that one million cells with an average of 1.

Of course, such decisions depend on the prognosis for the disease, the risk—benefit ratios for alternative treatments, and we use these reasonable numbers for illustrative purposes.

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There are many possible methods to quantitate the risk of a gene therapy protocol. These assays will evolve as our understanding of oncogenesis improves and other possible adverse events of gene therapy are revealed. We believe these quantitative risk assessments should be performed not just with current viral-based strategies but also with ones in the future that may include the use of potentially improved viral vectors, insulators, the use of targeted integration such as by phiC31 integrase or zinc finger nucleases ZFNs , and manipulated stem cells.

We envision two general types of assays to assess the oncogenic risk of a gene therapy protocol: cell-based and mouse-based. Cell-based assays, such as the 3T3 transformation assay, are well-established and standardized. A standard procedure to assess the oncogenic property of a gene, a gene variant, or a chemical compound is to determine the rate that it transforms 3T3 cells into cells that can grow in soft agar.

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A gene therapy protocol could be tested for its ability to transform 3T3 cells. Ideally, the protocol would not transform 3T3 cells above the background rate. Once that is achieved, the gene therapy protocol could be more rigorously evaluated by determining the rate it transforms 3T3 cells that have been further sensitized to transformation, such as by co-treatment with mutagenic chemicals or co-transformation with a weak oncogene. By using sensitized 3T3 cells, subtle differences between gene therapy protocols may be revealed.

The second way gene therapy protocols should be tested is using quantitative animal models. Ideally, one would like an animal model that in one to two years could accurately predict the oncogenic risk in a human for 50 years. As discussed above, standard mouse models are inadequate for this purpose, and such an ideal model does not currently exist. An alternative is to use mouse models that have been sensitized to tumor development cancer susceptibility model. For example, if one were interested in studying the oncogenic risk of a hematopoietic-based gene therapy protocol, murine hematopoietic cells deficient in p53 could be tested.

Ideally, one would find that even if million integration events a hypothetical number of events determined to be needed for clinical efficacy for that disease were transferred into p53 deficient cells, the latency to lymphoma development would not change. The protocol could be more rigorously tested by examining the latency to lymphoma after further sensitizing the cells by treatment with a chemical mutagen or co-transforming with a weak oncogene.

If in these models tumors were induced at a rate fold lower than that needed for clinical efficacy it would provide strong preclinical evidence that the protocol was safe for human trials. We have outlined one such strategy to test the safety of a gene therapy protocol in animals, but there are many different models one could use, and these different models may be more appropriate for different gene therapy protocols.

Establishing a uniform animal model for testing would be beneficial because it would allow different gene therapy protocols to be directly compared. Moreover, it would be useful to test the gene therapy protocols that have already been used in human trials to correlate the rate of transformation in an animal model to what was found in the human trials, thereby establishing a baseline for future studies.

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In summary, quantitative assays for safety should be developed. We have proposed potential cell-based and mouse-based assays, but it is likely that researchers can develop better quantitative cell- and animal-based models that more accurately assess the oncogenic risk specific to each gene therapy protocol.

We acknowledge that the studies outlined above set a high bar for gene therapy research. By setting such a bar, however, we will retain the trust of the patients, the public, and the funding agencies on which our work depends. If the measure of risk is defined on a per event basis, then to rationally balance risk—benefit, quantitative measures of efficacy per event must also be developed. To illustrate this principle, we will use hemophilia B factor IX deficiency as an example. If a particular vector could, on average, generate 10 picograms per event, then to achieve clinical efficacy one would expect 50 million events to be necessary.

If, on the other hand, another vector gave 10 times as much factor per average event, then fold fewer events would be necessary and this improvement in efficacy might also translate into improved safety.

A Look to Future Directions in Gene Therapy Research for Monogenic Diseases

May et al. It is likely that for each disease, different quantitative measures will have to be established, but once these measures are established, they will allow rational comparison of different vector systems and protocols. To improve the risk—benefit ratio of gene therapy, it is important to continue to develop ways to minimize the risk of insertional oncogenesis.

Four possible ways to minimize the risk are: 1 continued improvements in vector design, 2 buffer the genome from the effect of viral integrations, 3 develop ways to control transgene integration, or 4 develop ways to expand a small number of genetically characterized modified stem cells in vitro and then re-infuse these non-oncogenic cells into the patient.

In the two gene therapy trials that caused proliferative diseases, the investigators used a retroviral vector using the viral LTR to drive expression of the transgene. In the last several years, different vectors have been formulated, such as self-inactivating vectors and lentiviral vectors, which may have improved safety. In addition, improvements in safety may be gained by using tissue-specific promoters to drive transgene expression. These tissue-specific promoters may give increased transgene expression thereby increasing the efficacy per given event, and thus fewer events would be needed for clinical efficacy.

By using more specific promoters, more tightly regulated expression may be obtained that might also minimize the potential oncogenic properties that overexpression may entail. In retroviral and lentiviral vectors, however, the LTR would still be present and could still potentially act as an enhancer for neighboring genes. It is also possible that more specific promoters will not confer significant safety advantages. In the SCID and CGD trials, for example, insertional activation of a proto-oncogene occurred in a cell-type—specific manner and not in a heterologous cell type.

An alternative vector system is to use transposons as a nonviral delivery method. Transposon-based delivery systems have been shown to work in a wide variety of cell types, including stem cells [ 30 ]. In their current formulations, however, the integration events remain uncontrolled and thus may suffer the same problems of integration-based viral delivery systems. In fact, transposon-based insertions are being developed as a tool for cancer gene discovery [ 31 ].

An active but still preliminary area of research is to try to create hybrid transposases to target transposon integration to defined regions of the genome. Viral integrants under the control of LTR promoters can activate genes near the insertion site [ 9 , 12 , 32 ]. Buffering the genome surrounding the insertion site might be beneficial in minimizing such effects.

When insulators are incorporated into integrating retroviral vectors being developed for gene therapy purposes, position-effect variegation is decreased and the amount of transgene expressed is increased for each insertion [ 34 — 36 ]. It is hoped that insulators will protect the surrounding genome from insertional activation from retroviral LTRs. From the structural organization of such vectors, however, one would only expect a decrease but not an elimination of LTRs acting as enhancers on neighboring genes.


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Studies directly testing the degree to which insulators protect the surrounding genome from LTRs are ongoing, the publication of which is much anticipated. While insulators may protect the genome from the insertional activation of proto-oncogenes, they may be limited because they will not protect against the insertional inactivation of tumor suppressors by viral integrations.

Currently there are two possible methods being developed to target transgene integrations: the use of the phage C31 integrase and homologous recombination. In the natural phage life cycle, the temperate phage infects bacteria and integrates into the host genome. One issue is whether the expression of the integrase causes genomic instability.

A conceptually appealing way to treat monogenic diseases caused by small mutations is to use homologous recombination to correct the mutations while leaving the rest of the genome untouched. Recently, two strategies have been found that increase the rate of homologous recombination to levels that might be of therapeutic use: the use of rAAV and the use of DNA double-strand breaks DSB. In , Russell and Hirata reported that cells infected with rAAV underwent a high rate of homologous recombination [ 46 ].

Russell's group subsequently demonstrated that rAAV could correct a variety of lesions in a wide range of cell types [ 47 ]. The mechanism by which rAAV mediates high rates of gene targeting remains unclear but does seem to involve the cell's endogenous homologous recombination machinery.

Building on this work, the Russell group then used rAAV-mediated homologous recombination in mesenchymal stem cells to inactivate a dominant mutation in the collagen Col1A1 gene that causes osteogenesis imperfecta [ 48 ].

bbmpay.veritrans.co.id/campo-de-criptana-conocer-gente-nueva.php By inactivating the dominant mutation, they demonstrated that the cells had phenotypic correction of the underlying defect. Several issues remain with rAAV-mediated homologous recombination. To obtain recombination rates of useful magnitude, infection of cells with high multiplicity of infections is required, increasing the incidence of random integration.

In fact, most integration events will be nonhomologous rather than homologous. A study of these nonhomologous rAAV integration events showed that many were associated with chromosomal rearrangements and occurred in the control regions of genes [ 25 , 49 ]. The limitation of this approach is that endogenous genes do not contain recognition sites for I-SceI or other homing endonucleases. ZFNs have shown promise in being such a reagent. These nucleases are artificial proteins in which a zinc finger DNA binding domain is fused to the nuclease domain derived from the type IIS restriction enzyme FokI and were first developed by Chandrasegaran and his colleagues [ 51 , 52 ].

The first demonstration that ZFNs might be useful for gene therapy was in when Porteus and Baltimore demonstrated that model ZFNs could stimulate gene targeting by several-thousand—fold [ 44 ]. Urnov et al. These results provide optimism about the possibility of using ZFNs to stimulate homologous recombination for therapeutic purposes. One of the appeals of ZFNs is that they can be modified to recognize novel target sequences.

Theoretically it may be possible to make ZFNs to essentially any target sequence, but a current area of active research is to establish the best practical method to do this. Whether this relatively simple modular-assembly approach is good enough to make highly active and specific ZFNs or whether more complex selection-based approaches are needed remains to be determined. DSBs are known to be mutagenic and can lead to chromosomal translocations. Studies of ZFNs in model organisms and mammalian cells have demonstrated that they cause cytotoxicity by the creation of DSBs [ 44 , 54 ].

An additional potential problem is that the donor DNA can also integrate in a random fashion. While the donor can be engineered so it is promoter-less and enhancer-less to minimize insertional activation of nearby genes, the random integration of the donor could still potentially inactivate genes such as tumor suppressors. An approach to make gene therapy safer is to take advantage of the ability of stem cells to expand. In this scenario, gene transfer would occur in vitro. Individual or small pools of cells would be isolated, expanded, and characterized.