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23 Cards in this Set
- Front
- Back
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Gene Therapy
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addition of genetic material (DNA, coding mRNA, regulatory RNA) to cells/tissues to either:
(1) Provide a functional copy of a missing or defective gene: - especially suited to single gene defects and discrete, easily accessible populations of target cells e.g. hemophilia, ADA deficiency (2) Prevent the action of a defective or mis-regulated gene - Huntingtin expression in Huntington’s chorea or the whole extra chromosome 21 in Down’s syndrome (3) Protection against infection - “immunize” cells with antiviral genes to protect against eg HIV (4) Activate an immune response - addition of HLA genes, immune stimulatory cytokines, or artificial immune recognition proteins (eg CARs) to promote recognition of cancer cells, virus-infected cells (5) To kill cells, or stop them growing - addition of ‘suicide’ or toxic genes, cell cycle inhibitors, antiangiogenesis factors etc. to kill cancer cells, prevent vascular graft restenosis (6) To make cells grow - addition of pro-angiogenesis factors, |
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Vector Delivery Methods
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system to deliver the genetic material into cells.
- simply plasmid DNA, - DNA or RNA complexes, liposomes or “nanoparticles” - derived from viruses Can be either Ex Vivo or In Vivo |
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Ex Vivo
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cells removed from body, incubated with the vector to introduce the new genetic material, and the
modified cells returned Good for hematopoietic system e.g. T cells for ADA deficiency |
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In vivo
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(a) Localized delivery: use bronchoscope, vascular catheter, endoscope, sub-retinal, intra-cranial
injection etc. to target delivery to intended population (b) Systemic delivery: vector would be directly introduced into patient and would either target the specific cells, OR, it could deliver the gene non-specifically, but expression of the therapeutic gene would be restricted to the intended target cells. |
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Viral Vectors
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exploit highly efficient virus machinery to deliver genes, but without causing disease.
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Retroviral Vectors
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advantage that they integrate their genomes into the host genome, so allowing for longterm persistence and gene expression
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Lentiviral Vectors
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are a subset, derived from HIV-1 – very good at delivering genes to hematopoietic cells
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Adenoviral Vectors
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not permanent (can be good thing in certain applications), but easy to make in large amounts. However, can provoke immune reaction
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Adeno-associated Viral Vectors
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long lasting even though don’t integrate, seem to be nonimmunogenic, increasingly popular
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Hematopoietic Stem Cells
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good targets for gene therapy. They found in the bone marrow of adults, in fetal liver, and cord blood. They give rise to all types of blood and immune system cells and so are good targets for gene therapy. They proliferate/differentiate to produce millions of mature blood/immune cells, so that gene correction of HSC
and their progeny requires a permanent vector – retroviral/lentiviral |
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Severe Combined Immune Deficiency
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(SCID; bubble baby disease) is the genetic absence of a protective immune system. It’s a fatal disease, with infants dying from overwhelming infections. More than a dozen genes have been found to be cause human SCID
eg adenosine deaminase (ADA) and common cytokine receptor gamma chain (gC gene, X-linked SCID) |
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Treatment of Severe Combined Immunodeficiency
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Can be cured by BMT from an HLA-matched sibling; BMT from less well-matched donor has lower success rate. If gene therapy could target HSC, there is a good chance of success because a complete immune system may develop from just a few corrected HSC. And this is especially true of the corrected cells
have a selective survival or differentiation advantage, which is true for ADA and gC corrected cells. |
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Adenosine Deaminase Deficiency (ADA)
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is a defect in purine metabolism, leading to a build-up of toxic metabolites. It especially affects the T cells in the body. Treatment is PEG-ADA enzyme replacement therapy: bi-weekly I.M., can restore, sustain immunity, but expensive ($200-500,000/yr)
The first ADA deficiency gene therapy trial using retroviral vectors was started in 1990, giving gene-corrected autologous (“their own”) T lymphocytes to two girls suffering from this disease. Patient 1 received a total of 11 infusions, through Summer 1992. Her total T cell level and her level of modified T cells have remained essentially constant since then at approximately 20%. However, hard to determine if successful as still receiving PEG-ADA drug. More recently, ADA gene therapy trials using HSC, with patients receiving mild myeloblation by Busulfan – “makes space” in marrow. Also, no PEG-ADA given, that could blunt the selective advantage of the engineered cells. (Milan) |
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X-linked SCID
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lack common gamma chain (gc), defects in T, NK and B cells. Lethal by 4 months if untreated,survival 10 years under sterile conditions. Gene therapy of HSC using retroviral vector containing the normalgc gene, was initially viewed as a great success. by 2002: 13 patients treated, 11 showed benefit (Paris).
However, 2002- 2005, three patients developed leukemia due to retroviral vectors integrating near to the LMO-2 gene (known to be associated with early childhood leukemia). Suggests that the gene therapy retroviral vector itself inappropriately activated the LMO-2 gene. This is a risk of ectopic expression - ie randomly inserting a gene and promoter in a cell, where it can be in the “wrong” place. Newer vector designs reduce thechance of this happening, but it remains a risk. Risk/benefit of gene therapy for SCID? Overall, 9 of 11 treated patients showed immune restoration. 3 developed leukemia (1 died, 2 in remission). So cure rate by gene therapy was as good as results with bone |
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In Situ delivery of a vector
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Leber’s congenital amaurosis.
Inherited form of progressive blindness (blind by adolescence), caused by defective RPE65 gene, involved in pathway sending signals to brain. Maguire et al (Upenn) performed gene therapy by injecting a normal copy of the missing RPE65 gene carried by an AAV vector under the retina of affected patients. Treated 12 patients, ages 8-44. (Only one eye injected in patients.) All saw improvement in sight, better results in younger patients. |
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Gene Therapy for HIV
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knocking out the CCR5 gene
HIV needs both CD4 and CCR5 to enter its host cell, the CD4+ T cell. ~1% of the population have 2 copies of a defective CCR5 gene – CCR532 – and so do not have any CCR5 molecules on their cells. However, homozygotes are almost completely normal - EXCEPT - they are profoundly resistant to HIV/AIDS. |
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Proof of Principle
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Timothy Ray Brown, (the “Berlin Patient”) was cured of HIV following an HSC transplant from a CCR5-negative donor, as part of his treatment for leukemia. This has led to idea that if a patient’s own HSC could be made CCR5-negative, the engineered cells would then make HIV-resistant T cells in vivo, and provide an anti-HIV effect. This can be done by either RNA interference to reduce expression of CCR5 (interferes with translation and mRNA stability), or through zinc finger nucleases to knockout the CCR5 open reading frame.
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Zinc Finger Nucleases
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a zinc finger binds to 3 bp of DNA and can be engineered to recognize a specific DNA sequence. Linking fingers lengthens the DNA binding site (3 fingers = 9bp).Adding a nuclease domain creates the ZFN. They bind to a
DNA target sequence in pairs, and specifically cut the DNA at that site. The cell’s repair machinery fixes the double-stranded break using the nonhomologous end joining repair pathway – but this is very error prone, frequently causing small deletions or insertions that destroy the CCR5 open -reading frame. City of Hope/USC are developing a clinical trial using ZFNs to disrupt the CCR5 gene in HSC. First patients are expected to be AIDS lymphoma patients, where the gene therapy will be an add-on to the chemotherapy and autologous stem cell transplant the patients receive as a cancer therapy. |
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Gene Therapy for Cancer
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Cancer cells usually have genetic defects eg they over-express an oncogene, or lack expression of a tumor
suppressor gene. Strategies include: 1. Replace a missing tumor suppressor genes 2. Block expression of over-active oncogenes (eg use ZFNs or RNA interference) 3. Insert “suicide genes” into tumors 4. Insert genes to induce anti-tumor immune responses (eg IL-2, GM-CSF, CD80) 5. Express genes which impede tumor neo-vasculature (eg angiostatin) 6. Add chemotherapy resistance genes to HSC to allow chemotherapy dose intensification |
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Suicide Gene Therapy
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involves adding a suicide gene to tumors to make them sensitive to a drug e.g. Herpes Simplex Virus thymidine kinase (TK) - an enzyme that activates a non-toxic pro-drug (ganciclovir) to a cytotoxic compound. Basic idea is that although potentially all of the cells in the body will get exposed to the (nontoxic) pro-drug, only those cells that have received the TK gene will be killed by it.
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Chimeric Antigen Receptors (CARs)
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Idea is to engineer T cells (not the cancer cells) to force
them to recognize a cancer cell as foreign and kill it. CARs contain a recognition domain, usually derived from an antibody, and the intracellular cell signaling part of a molecule that activates T cells to kill their targets. Some cancer cell targets include CD19 on B cells. T cells are harvested from patient, engineered to express the CAR (eg using a lentiviral vector), cultured to expand them ex vivo, then returned to the patient. This has been successfully used to treat children with ALL at U Penn. |
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Basic Science challenges of CARs
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Basic science problems we’re working on:
- How to deliver or target the gene therapy vector (in vivo would be best) - How to stop problems caused by random integration of eg retroviral vectors - How to prevent overt immune reactions or inflammation against the vector - How to prevent the genetically engineered cells being lost due to immune response against the transgene |
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Clinical Challenges with CARs
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Clinical challenges:
- Complex production process and products are biological, not chemical/drugs - Patient-specific, rather than universal - Intellectual property issues complex - High cost for each treatment Will the therapy be of long-term benefit and cost-effective? (eg Lifetime anti-HIV drugs 500,000K) |
