Term
| what is gene therapy? how can it be done? |
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Definition
| the treatment/prevention of disease by gene transfer. introduction of genes into cells/gene expression or product manipulation/specific cell death induction. |
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Term
| at this point is therapy done on somatic or germ line cells? what about on a fetus? |
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Definition
| somatic, germ line is not allowed. gene therapy might be more effective in utero, but it isn't done due to ethical issues |
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Term
| what are common vectors for gene therapy? why? what is 100% necessary before using viruses however? |
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Definition
| viruses are self-replicating pieces of genetic material that can control host cell gene expression and target specific cells. pathogenic properties do need to be eliminated however |
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Term
| what diseases are classic targets of gene therapy? |
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Definition
| monogenic diseases such as CF, SCID, hemophilia, alpha-1-antitrypsin, ornithine transcarbamylase deficiency, diseases where there is one known gene causing the disease that could possibly be replaced. some non-monogenic diseases such as parkinson's are also being researched. |
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Term
| is cancer a target of gene therapy? |
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Definition
| yes, it is the most prevalent target currently |
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Term
| is infectious disease a target for gene therapy? |
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Definition
| yes HIV is such an example |
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Term
| what are some common approaches/strategies for gene therapy? |
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Definition
| provide a correct copy of a missing/defective gene, (good for monogenic disease). deliver a therapeutic protein, (either secreted or acting w/in a cell), disrupt mRNA to down-regulate expression of a gene/gene product. |
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Term
| what are some ways disrupt mRNA to down-regulate expression of a gene/gene product? |
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Definition
| mRNA transcription or translation can be blocked via anti-sense oligonucleotides/siRNA, ribozmyes can be used to target and cleave specific parts of RNA, or transcription factor decoys (oligonucleotides), can be used that bind transcription factors and prevent expression of target genes |
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Term
| how does antisense RNA therapy work? |
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Definition
| an oligonucleotide has a complementary specificity for some type of RNA, binds to it and the resulting double stranded RNA is degraded by RNAse H. the antisense RNA can be released and recycled |
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Term
| how does siRNA therapy work? |
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Definition
| small interfering RNA is double stranded when it enters the cell, it is cleaved by "dicer" and RISC, (RNA-induced silencing complex) unwinds it. the resulting single strand fragments bind to target RNA and thus singles it out for degradation by RNAse H. |
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Term
| how can cell death be induced by gene therapy? what kind of disease is this useful in? |
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Definition
| a gene can be transferred in whose product is toxic or a lytic virus can be targeted to a specific cell type -> both are useful in cancer therapy |
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Term
| what are intrabodies? how can they be used to prevent disease? |
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Definition
| intrabodies are intracellular antibodies that bind to a specific protein and inhibit its function |
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Term
| how can the immune response be manipulated in gene therapy of disease? what kinds of disease would this be useful for? |
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Definition
| cytokines can be introduced, apoptosis can be induced, antigens and be introduced to induce an immune response, all of which can be useful with cancer or infectious disease. much research is being done at the moment with antigens/cytokines |
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Term
| what is a common viral vectors for gene therapy? |
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Definition
| adenovirus, adeno-associated virus (AAV) which is a parvovirus that requires adenovirus or herpes simplex for replication (in absence of these, it cannot replicate), retrovirus (particularly lentivirus), HSV |
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Term
| what non-viral vectors are being assessed in terms of gene therapy? |
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Definition
| naked DNA/RNA via condensed particles or via carriers of DNA such as liposomes/polyethylenimines |
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Term
| how are lipids associated with DNA? |
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Definition
| a promotoer and Rx gene are placed in a plasmd and complexed with cationic lipids |
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Term
| how are retrovirus genes used to create Rx gene vectors? |
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Definition
| GAG, POL, and ENV are retrovirus packaging genes that can be taken out and combined with a therapeutic gene along with long terminal repeats and a packaging signal |
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Term
| how are adenoviruses made into Rx gene vectors? |
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Definition
| the replication gene is removed, the Rx gene replaces it |
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Term
| what is a safety concern with creating viral vectors? |
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Definition
| cross reactivity between the sequences being removed and those you want to keep leading to the virus regaining the ability to replicate |
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Term
| what are the 3 most common gene Rx vectors? |
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Definition
| adenovirus, retrovirus, naked/plasmid DNA |
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Term
| what are the 2 main approaches to gene therapy? what virus vectors are associated with each? |
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Definition
| in vivo/ex vivo. with in vivo, the gene is delievered to cells within the body and the vectors may need to be manipulated to target specific cells (adenovirus is the common vector here). with ex vivo, the patient's cells are removed, the gene is added and the cells are replaced (adenovirus is the common vector here and this approach is often used with leukemia) |
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Term
| what are considerations for designing gene therapy protocols? |
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Definition
| target cell specificity (vectors can be manipulated here), efficiency of transfer (% cells that recieve the gene), and immunogenicity, (immune response might make antibodies to the 2nd viral vector dose and clear it - affecting the efficiency of subsequent doses), magnitude & duration of response (whether Rx gene/containing cell is integrated/degraded), manufacturing, (expense, quality control, ease of production) and safety issues |
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Term
| what are safety issues in terms of the immune response to the viral vector? |
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Definition
| the immune response to an adenovirus can cause pt death, (as with jesse gelsinger who was under therapy to correct ornithine transcarbamylase deficiency). elevated IL-6 can lead to fever, myalgia, liver damage, resp distress syndrome, and mult. organ failure (massive systemic inflammatory response triggered by vector). |
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Term
| what is a safety issue with gene therapy in terms of insertional mutagenesis? what is an example of this being problematic? |
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Definition
| random integration by a viral vector might inactivate tumor suppressor genes. there was an X-linked SCID gene therapy program using an ex-vivo approach to correct the IL2RG gamma chain gene in cells that were reintroduced to pt's bone marrow. 8 of 10 pts gained gamma chain positive T cells w/normal functioning as well as some corrected NK & B cells, but 2 pts got T cell leukemia b/c integration of the Rx gene occured near a proto-oncogene promotor (LMO2 gene), leading to its increased expression = leukemia |
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Term
| what are advantages of some viruses as vectors? |
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Definition
| viruses can be designed to target specific cells, integration allows for prolonged expression of the gene (vector dependent), gene transfer is relatively efficient, it works both in and ex vivo, and transduces both proliferating/non-proliferating cells (vector dependent) |
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Term
| what are disadvantages of viruses as vectors? |
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Definition
| potential to cause disease w/replication competent vectors, potential to stimulate an immune rxn via immune-mediated pathology or less effective subsequent doses, concerns regarding random integration via insertional mutagenesis, difficulty in manufacturing, and smaller vectors limit the insert size |
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Term
| what are non-integrating vectors? what are integrating vectors? |
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Definition
| adenovirus, HSV are non-integrating vectors. retroviruses and adeno-associated viruses are are integrating vectors. |
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Term
| what are some efficient transfection vectors? inefficient? |
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Definition
| adenovirus (in/ex vivo), retrovirus (ex vivo) and AAV (in vivo) are efficient. retrovirus (in vivo) and AAV (ex vivo) are inefficient transfectors. |
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Term
| what viral vectors have low immunigenicity? high? |
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Definition
| retrovirus, AAV have low immunogenicity. adenovirus has high immunogenicity. |
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Term
| what are pros/cons of retrovirus as a viral vector? |
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Definition
| retrovirus cons: efficent DNA transfer (ex vivo), stable expression, low immunogenicity. cons: transfer to dividng cells, DNA integration (safety), DNA transfer size is limited |
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Term
| what are pros/cons of adenovirus as a viral vector? |
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Definition
| adenovirus pros: efficient DNA transfer edp to nondividing cells. cons: transient expression, host immune response, limited insert size |
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Term
| what are pros/cons of AAV as a viral vector? |
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Definition
| AAV pros: prolonged expression, transfer to nondividing cells, low immunogenicity. cons: limited insert size, DNA integration, (safety) |
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Term
| what are pros/cons of naked DNA as a viral vector? |
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Definition
| pros: safe, easy, no size limit, low immunogenicity. cons: low efficiency, transient expression, hard to target specific cells |
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Term
| what are pros/cons of liposome-DNA as a viral vector? |
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Definition
| pros: safe, ease of production, no DNA size limit, efficient transfer (ex vivo), low immunogenicity. cons: low efficiency (in vivo), transient expresion, hard to target specific cells. |
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Term
| what are characteristics of an "ideal vector"? |
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Definition
| good target cell specificity, (esp w/metastasized CA), efficent gene transfer, (w/both dividing+non-dividing cells), expressed gene has desired effect (better if expression can be regulated), repeat dosing is safe & effective (not immunogenic/specific site integration), accomodates large genes, easy to produce |
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Term
| what are ways vectors can be manipulated to target specific cells? |
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Definition
| alter the receptor binding site of the viral vectors, use viral vectors that naturally target your target cell, (HSV -> neurons), incorporate ligands for cellular receptors into liposomes, and manipulate vectors so that gene expression occurs only in specific cell types (requires presence of absence of a specific host factor) |
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Term
| how can safety issues be dealt with in terms of viral vectors? |
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Definition
| virulence genes should be removed, minimize/eliminate the immune response (use non-viral vectors or remove viral antigens), avoid insertional mutagenesis (use a vector that binds at a known site/direct it), and eliminate replication competent viruses |
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Term
| what are advantages of in utero gene therapy? |
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Definition
| the defect can be corrected before the disorder manifests itself, introducing the gene to stem cells may allow gene delivery into multiple cell types upon stem cell differentiation (requires vectors that can integrate and be maintained during cell division b/c long term expression of transferred gene is necessary), and gene therapy in an immature immune system may prevent an immune response to the vector/gene product (vector/gene product might be considered "self", tolerance could be developed and re-administration of the vector to the child would be safer/more efficient) |
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Term
| what are concerns for in utero gene therapy? |
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Definition
| long-term safety issues (animal studies may not be enough, insertational mutagenesis could lead to other defects/malignancies), the possibily of unintentional integration of the gene into germ cells |
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Term
| when might in utero gene therapy be justified? what are some diseases considered? |
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Definition
| if life-threatening disorders with known organ damage occur early in life or when there is lack of effective alt tx. diseased considered include hemophilia B, ornithine transcarbamylases deficiency, phenylketonuria, fragile X syndrome, and familial hypercholesteremia |
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Term
| what inherited disease causing blindness in the 30s-40s due to retinal degeneration is being investigated for gene therapy? what does the gene therapy involve? |
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Definition
| leber's congenital amaurosis which involves a mutation in the RPE65 gene which is needed for metabolism of vit A in the eye, (used by rods/cones to make pigment). it involves an in-vivo approach into the retina which has been moderately successful. |
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Term
| what is X-linked adrenoleukodystrophy, (ADL)? how can it be treated with gene therapy? |
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Definition
| a fatal demyelinating disease caused by mutations in the ABCD1 gene where peroxisomes that are supposed to degrade VLCFAs don't and the maintenance of myelin is disrupted. current gene therapy consists of taking CD34 mononuclear blood cells, infecting them with lentiviruses containing ABCD1 cDNA. bone marrow cells in pts are then depleted and re-populated with the treated cells. this has been shown to work |
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