Gene Therapies. Josh Crockett, Lexi Trujillo, Caitlin Halliday
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1 Gene Therapies Josh Crockett, Lexi Trujillo, Caitlin Halliday
2 Gene Therapy Main objective: create carriers that can envelope and deliver genetic materials into target tissues (like cancerous cells) in order to treat diseases at a genetic level. Genes used: TSG (induces apoptosis) or anti-oncogenes (suppresses proliferation)
3 Gene Therapy
4 Gene Therapy Delivery Mechanisms Viral Vectors: Modified to be unable to reproduce, still able to transfer genetic material. Pros: High gene transfer capability. Cons: Can t transfer high quantity of genetic material, still a virus (immunogenicity) Cationic Vectors: Interact with negatively charged DNA, but total overall charge remains positive to interact with negatively charged cell membrane (endocytosis). Pros: Less efficient than viral vectors in gene transfer. Cons: Safer, simpler preparation, higher gene encapsulation ability.
5 Viral Vectors: Retrovirus HIV (AIDS) Small RNA viruses with a DNA intermediate. Inserts into host genome and produces therapeutic proteins Mostly infects actively-dividing cells Even rapidly-growing tumors have non-dividing cells (G0 phase), so Lentivirus is used to infect these.
6 Viral Vectors: Adenovirus Actual virus causes symptoms similar to common cold. Double-stranded DNA virus, infects dividing and non-dividing cells. Viral E1/ E2 gene replaced with therapeutic gene. Transfection is temporary as viral genome isn t permanently integrated into host genome. More efficient in immunodeficient hosts Administering immunosuppressants is usually undesirable because immune system is often used to fight tumors
7 Viral Vectors: Adeno-associated Virus Single-stranded DNA virus, encodes for Rep and Cap proteins which are replaced with therapeutic genes. Pros: Infects both dividing and non-dividing cells. Repetitive treatments unnecessary. Low risk of viral infection (wild type has no pathological effects in humans). Cons Transfection ability is less than that of adenovirus. Helper virus required for AAV production.
8 Viral Vectors: Herpes Simplex Virus Large, double strand virus Naturally infects mucus membranes and remains latent in neuronal cells Allows for delivery to brain tumors Used to deliver granulocyte macrophage colony-stimulating factor and IL-2 into mice tumors. Allows for largest delivery capacity of all viral vectors. Can deliver multiple genes simultaneously. HSV-tk and cytosine deaminase delivered with ganciclovir and 5-fluorocytosine delivered to malignant glioma
9 Viral Vectors: Pox Virus Double stranded DNA virus used in vaccines Infects dividing and non-dividing cells High capacity for carrying foreign DNA Used to deliver cytokines Low toxicity
10 Cationic Vectors: Polymers Any synthetic cationic polymer which can complex with DNA Many modifications can be made: Molecular weight, ligand attachment, branching Poly(ethylenimine) (PEI) Poly(l-lysine) (PLL) Rapidly cleared from circulation Transfection requires co-application of chloroquine Applied wth PEG coating (increases transfection and circulating half-life) and targeting ligands
11 Cationic Vectors: Peptides Contain positively charged amino acids which enable them to condense DNA. Efficiency depends on hydrophobic portion (aggregation and endosomal escape) DNA release into cytoplasm is improved by addition of cytosine particles into the peptide backbone. In vivo action is still under investigation. Transfection less effective in mice than PEI or liposomal vectors.
12 Cationic Vectors: Liposomes Positively charged lipids at physiological ph complex with positively-charged DNA = Lipoplex DNA is layered between liposomal particles. Poor stability, must be administered immediately Most common lipids used Cholesterol Dioleylphosphatidylethanolamine (DOPE) Enter cells through endocytosis or fusion. Conformational changes trigger release of encapsulated DNA into cytoplasm.
13 Types of Gene Therapy Immunotherapy Oncolytic virotherapy Gene transfer
14 Immunotherapy Cancer Vaccines: Vaccines are substances put into the body to start an immune response against certain diseases. We usually think of them as being given to healthy people to help prevent infections. But some vaccines can help prevent or treat cancer. 1. Cancer cells are grown in vitro. 2. They are engineered by the addition of genes to create a larger immune response in the body. 3. The cells are killed and injected into the patient to induce an immune response.
15 Oncolytic Virotherapy Oncolytic virotherapy uses viral particles that replicate within the cancer cell to cause cell death. Oncolytic viruses are therapeutically useful anticancer viruses that will selectively infect and damage cancerous tissues without causing harm to normal tissues
16 Oncolytic Virotherapy Each virus has a specific cellular tropism that determines which tissues are preferentially infected. Oncolytic vectors are designed to infect cancer cells and induce cell death through the propagation of the virus, expression of cytotoxic proteins and cell lysis. In addition to the killing of infected cells, oncolytic viruses can mediate the killing of uninfected cancer cells by indirect mechanisms such as the destruction of tumor blood vessels or the amplification of specific anticancer immune responses.
17 Oncolytic Virotherapy The viruses need to be specific in order to kill cancer cells. Some viruses already have a preference to cancer cells due to an attraction of certain cell markers on the surface of cancer cells and some viruses can be modified to have a preference for cancer cells. Surface markers such as EGF receptor, Her2-neu, Folate receptor, Prostate Specific Membrane Antigen and CD20, and nuclear transcription factors PSA, htert, COX-2, osteocalcin expressed selectively by tumor cells can be targeted by using them as receptors for virus entry or as essential cofactors for viral gene expression.
18 Gene Transfer The introduction of new genes into a cancerous cell or the surrounding tissue to cause cell death or slow the growth of the cancer.
19 Gene Transfer TNFerade This agent is a replication incompetent adenoviral vector that delivers the tumor necrosis factor-α (TNF-α) gene under the transcriptional control of a radiation inducible promoter. TNFerade is injected into tumors and then is activated by radiation therapy. Once activated by radiation, the cell produces TNF-α which when coupled with the radiation therapy promotes death in the cancerous cells as well as some cells in the area.
20 Clinical Trials Phasing Phase 1: Finding toxic or safe levels of the drug & side effects. Phase 2: What type of cancer to treat with the drug, how to manage side effects Phase 3: comparison studies to old treatment regimen, or addition to standard treatment Trials can be in more than one phase Phase ½ or ⅔ Multi-arm Multi-stage these trials are still random but compare several treatments at once (multi- arm) if one treatment isn't working as well as others or one is more beneficial they may stop or add treatment groups (MAMS)
21 Gene Therapy and Radioactive Iodine in Recurrent Prostate Cancer Used in men 18+ who did not respond to external beam radiation This phase I trial is studying the side effects and best dose of gene therapy given together with radioactive iodine in treating patients with locally recurrent prostate cancer that did not respond to external-beam radiation therapy. Radioactive drugs, such as radioactive iodine, may carry radiation directly to tumor cells and not harm normal cells. Placing a gene called type 5 adenovirus (Ad5)-cytomegalovirus (CMV)-sodium-iodide symporter (NIS) in prostate cancer cells may help the prostate cells take in more radioactive iodine and thus kill the cancer cells. Drugs, such as liothyronine sodium, may protect the thyroid from the side effects of radioactive iodine. cdrid=618542&version=healthprofessional&protocolsearchid=
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23 Gene Therapy of Pancreatic Ductal Adenocarcinoma (TherGAP) Patients must have a non resectable pancreatic adenocarcinoma Near 85% of patients with pancreatic adenocarcinoma are diagnosed with a locally advanced and/or metastatic unresectable tumor. In these patients chemotherapy (such as gemcitabine) is given as a palliative therapy. Aim of the present study is to evaluate the feasibility, tolerance and antitumor effect of repeated intratumoral injection of a gene therapy product (with antitumor and chemo sensitizing effects) combined with gemcitabine in patients with unresectable pancreatic carcinoma. This is a gene therapy open nonrandomized phase I trial for advanced and/or metastatic pancreatic cancer patients. The protocol is based on the administration of increasing doses of a plasmid DNA that encodes two genes which exhibit complementary therapeutic effects. Both transgenes induce an antitumor bystander effect and render gemcitabine treatment more efficient. Intratumor injections of the gene therapy product (CYL-02) will be performed by transgastric or transduodenal route under endoscopic ultrasound guidance. Each injection will be followed standard gemcitabine IV administration every week (1000 mg/m2). Two intratumor injections of a same dose of CYL-02 will be administered at one month interval. Four increasing doses (125 µg, 250 µg, 500 µg and 1 mg) will be tested by group of 6 patients.
24 Randomized Trial of Suicide Gene Therapy and Prostate Cancer (ReCAP) This is a randomized phase 2/3, controlled trial that will test the hypothesis that replicationcompetent adenovirus-mediated suicide gene therapy in combination with 80 Gy intensity modulated radiotherapy (IMRT) will improve freedom from failure (FFF) relative to 80 Gy IMRT alone in patients with newly-diagnosed prostate cancer with an intermediate-risk profile has two arms: Arm 1- Gene Therapy + IMRT Arm 2- IMRT This study is now COMPLETE!
25 Randomized Trial of Suicide Gene Therapy and Prostate Cancer (ReCAP) Arm 1- Gene Therapy + IMRT more flu like symptoms, more transaminitis (can indicate liver damage), low white blood cell count, low platelets after 2 years: 33% biopsies came back positive Arm 2- IMRT after 2 years: 58% biopsies came back positive
26 Long-Term Follow up of Recipient of Gene Transfer Research This study is actively recruiting patients in order to be considered for this study the patient must have received retroviral gene therapy in the past. This study is a long term follow up or survey of a patient's first clinical trial first 5 years patient will have blood samples drawn next 10 years patient will have questionnaire to fill out The goal of this clinical research study is to collect information about the long-term safety of gene transfer therapy. This study is also designed to create a database of medical information about patients who have received gene transfer therapy, in order for researchers to track and review the long-term safety and any effects (good or bad) of gene transfer therapy.
27 Risks of Gene Therapy Immune response: the immune system may cause unwanted immune response to the new virus immune response could lead in unwanted inflammation, or death. Jesse Gelsinger s Death lead to the halt of gene therapies in the U.S. until safety measures could be determined. Immunosuppressants are now used in conjunction with gene therapy less potent vectors
28 Risks of Gene Therapy Target the wrong cells: it is a virus it may affect more than one cell, potentially infecting a healthy cell later in life it may mutate and cause other diseases or cancers this was seen in X- linked Severe Combined Immunodeficiency (X-SCID) gene therapy after gene therapy was complete some patients developed leukemia must not mutate to reproductive cells of the patient, if so the gene therapy could pass on to offspring after treatment Must be a working version of the gene delivered within the vector if gene becomes mutated to be overexpressed
29 Costs of Gene Therapy Because gene therapies are so personalized these costly but potentially life saving therapies may not be available to everyone These treatments must be synthesized by pharmaceutical companies and be cost effective Must then pass FDA approved clinical trials Alliance for Cancer Gene Therapy ACGT is the only public charity in the nation exclusively funding cancer cell and gene therapy research
30 References Cancer research UK. October 21, April 20, Cross, D., & Burmester, J. K. (2006). Gene Therapy for Cancer Treatment: Past, Present and Future. Clinical Medicine & Research, 4(3), doi: /cmr Freytag SO, Stricker H, Lu M, et al. Prospective Randomized Phase 2 Trial of Intensity Modulated Radiation Therapy With or Without Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy in Intermediate-Risk Prostate Cancer.International journal of radiation oncology, biology, physics. 2014;89(2): doi: /j.ijrobp Genetic Science Learning Center (2014, June 22) Challenges in Gene Therapy?. Learn.Genetics. Retrieved April 21, 2016, from Sibbald B. Death but one unintended consequence of gene-therapy trial.cmaj: Canadian Medical Association Journal. 2001; 164(11):1612. Russell, S. J., Peng, K., & Bell, J. C. (2012). Oncolytic virotherapy. Nat Biotechnol Nature Biotechnology, 30(7), doi: /nbt
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