gene therapy for the treatmen of cancer

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Gene Therapy for the Treatment of Cancer: From Laboratory to Bedside G Jeni Christi A G851130321

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Page 1: Gene therapy for the treatmen of cancer

Gene Therapy for the Treatment of Cancer: From Laboratory to Bedside

G Jeni Christi AG851130321

Page 2: Gene therapy for the treatmen of cancer

Gene therapy is an experimental treatment that involves inserting genetic material into your cells to give them a new

function or restore a missing function, as cancer may be caused by damaged or missing genes.

Gene therapy is designed to modify cancer cells at the molecular level and replace a missing or bad gene with a healthy one. The new gene is delivered to the target cell via a ‘vector,’ which is usually an inactive virus or

liposome.

Gene Therapy

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Schematic diagram of gene transfer therapy

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Several approaches:1.Modification of tumor cells2.Sensitization of normal

tissues or tumor cells3.Modulation of tumor

invasiveness4.Enhancement of the

antitumor immune response

STRATEGIES OF GENE TRANSFER FOR THE TREATMENT OF CANCER

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MODIFICATION OF TUMOR CELLS

Repairing genetic defects believed to be responsible for tumoral proliferation, for example, by restoring genes controlling cellular division or that induce programmed cell death (apoptosis).

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In theory, gene therapy could be used to replace an inactive gene with an active one or to neutralize an abnormal function gained by a mutated gene. Inactivation of regulatory genes frequently induces tumor cell growth.

For example, mutations of the p53 gene have been identified in numerous tumor models. The exact role of the wild p53 gene has not been totally identified, but its product suppresses the expression of genes involved in cellular proliferation and may induce proapoptotic genes.

Restoration of the activity of the p53 gene in p53-mutated or-deficient tumors may stop uncontrolled cell growth or induce apoptosis. Several strategies using adenoviral, retroviral, or nonviral p53 gene transfer have tested this hypothesis in preclinical tumor models. Clinical trials have demonstrated the potency of p53 gene transfer in hepatocellular, head and neck, and lung carcinomas.

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Schematic representation of p53 transfection and consequent apoptosis induction in targeted cancerous cells

El-Aneed 2004

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Oncogenes or their products are the targets of several therapeutic strategies that aim at restoring their normal function either to stop anarchic cell growth or to induce apoptosis. Oncogenes from the ras family (H-ras, N-ras, and K-ras) are activated by a simple point mutation. It has been possible in a lung cancer model to block the mRNA of a mutated K-ras gene and subsequently prevent the secretion of the altered protein and delay tumor cell growth in vitro and in vivo.

Similar approaches have been tested to block the effects of fos oncogene in a murine model of mammary tumor using a retroviral vector.

Other methods using ribozymes, antisense RNA, or intracellular anti bodies have shown promising results, but are too preliminary to envision clinical applications in the near future.

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Numerous difficulties must be overcome before tumor correction strategies can be successful in a clinical trial, for example:

1. It will be necessary to get a corrective gene into an extremely high proportion of malignant cells, although it has been suggested that there is some form of uncharacterized ‘‘bystander’’ effect on nontransduced tumor.

2. Targeting metastases will usually be necessary. 3. Correction of a single defect may be inadequate to actually

kill the tumor cells, leaving instead a collection of ‘‘n-1 cells’’ (where n is the number of mutations required for malignancy to occur) capable of undergoing another mutation to restart the malignant process.

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SENSITIZATION OF NORMAL TISSUES OR TUMOR CELLS

Introducing into the tumor cells genes encoding for an enzyme that can transform a nontoxic prodrug into an active drug, or by introducing genes into normal tissues that can protect them against the effects of antitumor toxic drugs.

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SENSITIZATION OF NORMAL TISSUES OR TUMOR CELLS

A. Prodrug-metabolizing EnzymeB. Cytotoxic Drug-resistance

Gene Transfer

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A. Prodrug-metabolizing Enzyme

The concept is that the gene encoding the PDME is expressed in the cancer cell and metabolizes a small molecule to an active moiety, which then kills the tumor cell directly. The molecule may also diffuse either through intercellular gap junctions or in the extra cellular space and destroy adjacent tumor cells. In this way, transduction of even a small proportion of tumor cells can produce a large ‘‘bystander’’ effect on adjacent tumor tissue.The major limitation of PDME—that it requires local inoculation of a tumor with the vector encoding the gene—does not represent a major disadvantage.

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Brain tumors were an attractive initial target for PDME gene therapy. Because the tumors seldom metastasize, the goal of the therapy is the local eradication of the tumor.

More recently, a study has been performed on patients with retinoblastoma, which is also a highly localized tumor that is conventionally treated by enucleation and/or chemoradiotherapy. Enucleation is obviously disabling and deforming, and if the tumor is bilateral it leads to blindness.

Other suicide gene therapies are being evaluated. Among the most developed of these is the cytosine deaminase system, which converts fluorocytosine to fluorourosil. There are, however, concerns that this suicide system may be less potent than the Tk-ganciclovir prodrug system.

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PDME has also effectively been used as a means of controlling T cell therapies. For example, graft vs. host disease (GvHD) may occur when donor T cells are given to patients after allogeneic stem cell transplantation in an effort to treat tumor relapse (graft vs. tumor effect) or posttransplant infections. Several groups have infused donor T cells transduced with the HSV-Tk gene and reported successful abrogation of GvHD after treatment with ganciclovir.

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El-Aneed 2004

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B. Cytotoxic Drug-resistance Gene Transfer

The concept is that giving more of a cytotoxic drug over a longer period will cure a higher proportion of patients. By increasing the therapeutic index in this way, it is hoped that more drug can be administered and a higher percentage of patients cured.

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2 major problems with using transfer of drugresistance genes: 1. The lack of targeting of current vectors means that they

may transduce malignant cells and normal cells, and therefore increase the resistance of both to the cytotoxic drug.

2. Although it may be possible to protect a significant proportion of marrow stem cells, secondary toxicities to other organ systems such as skin, lung, and gut will rapidly become evident as doses are escalated because these tissues are much less readily protected than hematopoietic stem cells.

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Nooter & Stoter 1996

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ANTIANGIOGENESIS GENE THERAPY

Delivering genes that can inhibit the growth of new blood vessels to impede nutrient supply to the tumor cells (inhibition of neoangiogenesis)

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Tandle et al. 2004

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For example, endostatin, a 20-kd fragment of collagen XVIII can efficiently block angiogenesis, but the recombinant protein is difficult and expensive to produce and is somewhat unstable. Delivery of endostatin in murine tumor models by several different vector systems has been able to overcome this limitation and has proved extremely promising. Similarly, angiostatin, a fragment of plasminogen, also functions as a large molecule inhibitor of vessel growth and impedes metastastic tumors. This too can be transferred (e.g., by adeno-associated virus vector) to produce benefit in animal models of malignant brain tumors.

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GENE MODIFICATION OF THE IMMUNE RESPONSE

Inducing the recognition of tumor cells by the host’s immune system or by enhancing the cytotoxic function of immune effectors.

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Schematic diagram of immunotherapy

Cross & Burmester 2006

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GENE MODIFICATION OF THE IMMUNE RESPONSE

A. Tumor EscapeB. Antigen Presentation DefectC. T Cell DefectD. Microenvironment AbnormalitiesE. Clinica Application of Immuno-gene

TherapiesF. DC Clinical VaccinesG. Nucleic Acid VaccinesH. Future Prespective for

Immunotherapy in Cancers

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A. Tumor Escape

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B. Antigen Presentation DefectSeveral immunotherapy strategies have been conceived

to overcome these defects. Earlier murine models had shown that an increased expression of MHC class I molecules decreased tumorigenicity, due to enhanced antigen presentation to CD8 cytotoxic T lymphocytes (CTLs). Tumor immunogenicity has also been increased by gene transfer of both allogeneic MHC class I and II molecules, thus demonstrating the relevance of both CD8 cytotoxic and CD4 helper T cells in enhancing systemic immunity against cancer. Increased expression of MHC molecules can also be obtained indirectly by transducing tumor cells with cytokines able to induce MHC molecule up-regulation on the cell surface: examples of cytokines with this property are interferon- (IFN-), IL-4, and Tumor Necrosis Factor- (TNF-).

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Once the T cell receptor (TcR) has specifically interacted with the epitope, costimulatory signals induce a T cell response and prevent anergy. A number of such signals have been identified of which the B7 family members, such as B7.1 (CD80) and B7.2 (CD86), are among the best known. These molecules are expressed on the antigen-presenting cell (APC) surface and bind to their cognate receptor, CD28, on the responding T cell. Other costimulatory molecules, such as intercellular adhesion molecules and leukocyte function-associated antigens are also important. This important role of CD80/86-CD28 interaction in T cell activation made B7 genes an appealing target for gene transfer into tumor cells.

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Once the T cell receptor (TcR) has specifically interacted with the epitope, costimulatory signals induce a T cell response and prevent anergy. A number of such signals have been identified of which the B7 family members, such as B7.1 (CD80) and B7.2 (CD86), are among the best known. These molecules are expressed on the antigen-presenting cell (APC) surface and bind to their cognate receptor, CD28, on the responding T cell. Other costimulatory molecules, such as intercellular adhesion molecules and leukocyte function-associated antigens are also important. This important role of CD80/86-CD28 interaction in T cell activation made B7 genes an appealing target for gene transfer into tumor cells.

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C. T Cell DefectMultiple strategies have been used to overcome T cell

defects. Most commonly, tumor cells are transduced with genes encoding cytokines able to re-create an optimal microenvironment for T cells recruitment, activation, and expansion. Several cytokines can be used alone or in various combinations to enhance antitumor T cell-mediated immunity: recruitment of cytotoxic CD8 T cells able to recognize tumor-specific antigens is observed with IL-4, whereas CD4 T cell recruitment is favored by TNF and IL-7. Costimulatory surface molecules such as B7.1 or CD40L may serve as accessory signals in the T cell activation process and prevent/overcome the T cell anergy induced by tumors

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D. Microenvironment AbnormalitiesTumors can secrete substances able to induce

immunosuppression. The best characterized of these are TGF-, IL-10, and vascular endothelial growth factor (VEGF). TGF- affects CTL function, inhibiting production of Th1 cytokines (especially IL-12), down-regulating surface expression of IL- 2 receptors on T cells, inhibiting antigen presentation on MHC class II molecules and decreasing surface expression of costimulatory and adhesion molecules. IL-10 antisense gene transduction can restore immunogenicity when tumor cells produce high amounts of IL-10. VEGF serves to promote tumor angiogenesis and inhibit DC differentiation.

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E. Clinical Applications of Immune-gene Therapies

1. Gene-modified Autologus and Allogeneic Tumor Cells

2. Cancer Therapy with Gene-modified T Cells

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1. Gene-modified Autologus and Allogeneic Tumor Cells

Several clinical applications in humans have been reported using manipulated autologous cancer cells (93). When transduced autologous tumor cell lines cannot be obtained (because the tumor is not accessible, because the tumor cells do not grow ex vivo, or because gene delivery is difficult), an immunogenic allogeneic tumor cell line can be a valid alternative. This approach unfortunately has several limitations: (1) The tumor antigens present on the autologous tumor population may be absent in the tumor cell line; (2) the antigen may be presented on a mismatchedMHCmolecule and, in the absence of cross-priming of host lymphocytes, may fail to be recognized by host T and cells; (3) the tumor antigen may not contain peptides capable of being presented by host APC so an immunogenic allogeneic tumor in one individual may be nonimmunogenic in a second patient with a different human leucocyte antigen (HLA) type.

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2. Cancer Therapy with Gene-modified T Cells

a. Protecting T cells Against Tumor-induced Down Regulation.

b. Chimeric T Cells for Tumor Therapy

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a. Protecting T cells Against Tumor-induced Down Regulation.

Clinical studies using CTL against EBV-related malignancies are promising and there have been tumor responses, particularly in patients with PTLD. Nonetheless, no patient with aggressive relapsed EBV Hodgkin’s disease has been cured using EBV-specific CTL. This may be due to a lack of specificity of the EBV-specific CTL for the immunosubdominant LMP1 and LMP2 antigens that are all present on the Hodgkin tumor cells. In addition, the tumor secretes immunosuppressive cytokines and chemokines, which affect CTL function and APC activity. Gene transfer can be used to overcome both types of problems. By using dendritic cells transduced with adenoviral vectors encoding either LMP2 or a mutated LMP1, it is possible to generate CTL that have high cytolytic activity in vitro to LMP2- or LMP1-positive targets when compared with conventional EBV-CTL.

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b. Chimeric T Cells for Tumor Therapy

Primary T cells genetically modified to express chimeric receptors derived from antibodies and specific for tumor or viral antigens have considerable therapeutic potential. Chimeric T cell receptors allows the recognition specificity of T lymphocytes to extend beyond classical T cell epitopes by transducing cells with genes that encode the variable domain of a tumor-specific monoclonal antibody (MAb) single-chain fragment (ScFv) joined to a cytoplasmic signaling domain. This strategy can therefore be applied to every malignancy that expresses a tumor-associated antigen for which an MAb exists

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F. DC Clinical Vaccines

To overcome the antigen presentation defects in antitumor immune recognition, DCs can themselves be manipulated ex vivo and used as cancer vaccines, mainly acting by priming naive T cells. DCs can be induced to present tumor antigens by several strategies, including feeding with tumor cell lysates and apoptotic bodies or by using tumor-derived RNA or making DC–tumor cell hybrids. Specific tumor antigen gene transfer into DC using viral or nonviral vectors is also possible

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G. Nucleic Acid VaccinesNucleic acid vaccines induce an immune response

targeted against a protein expressed in vivo subsequent to the administration of its encoding DNA or RNA.

Nucleotide-based vaccines have several advantages over proteins and peptides.1. Provide prolonged antigen expression that can continuously

stimulate the immune system, probably through an intracellular antigenic reservoir, resistant to antibody-mediated clearance.

2. Nucleotide vaccination leads to antigen processing through both the endogenous and exogenous pathways, so that specific CTL and helper T cells can be recruited.

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H. Future Prespectives for immunotherapy in Cancer

Tumors possess multiple and powerful strategies to escapeimmune surveillance and can develop new immune evasionmechanisms during disease progression.

Although no ‘‘breakthrough’’ clinical success has been reported, a better understanding of immune evasion strategies and the availability of improved technologies of immune manipulation have opened the way for real immunotherapies of cancer that should ultimately deliver on the dreams of the 19th and 20th centuries.

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Conclusion Improvement of gene transfer for the treatment of cancer certainly relies on 4 major steps: 1. simplification of gene transfer protocols, still too complex to

implement in a clinical environment; 2. controllable, tissue-specific regulation of transgene

expression;3. progress in the understanding of carcinogenesis mechanisms

to improve therapeutic strategies; 4. improvement in the methodology of clinical trials, including

optimal choice of the patient population, and monitoring of tumor and immune responses, within the tight frame of regulatory and cost-related issues.

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1. Rousseau RF, Biagi E, Pule M & Brenner MK. 2004. Gene Therapy for the Treatment of Cancer: From Laboratory to Bedside. Gene and Cell Therapy. New York : Marcel Dekker, Inc.

2. Noorten K, & Stoter G. 1996. Molecular Mechanisms of Multidrug Resistance in Cancer Chemotherapy. Path. Res. Pract. 192, 768-780 (1996).

3. Cross D, & Burmester JK. 2006. Gene Therapy for Cancer Treatment: Past, Present and Future. Clinical Medicine & Research Volume 4, Number 3: 218-227.

4. El-Alneed A. 2004. Current Strategies in Cancer Gene Therapy. European Journal of Pharmacology 498 (2004) 1-8.

5. Tandle A, Blazer DG, & Libutti SK. 2004. Antiangiogenic Gene Therapy of Cancer: Recent Development. Journal of Translational Medicine 2004, 2:22 doi:10.1186/1479-5876-2-22.

References

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Arigatoo Gozaima

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