Lec 4

Adeno-associated viruses as vectors for gene therapy: 

Adeno-associated viruses as vectors for gene therapy Adenovirus Smaller viruses that accompany adenoviral infection parvovirus family Non-pathogenic virus Needs adenovirus as helper for replication (naturally defective virus) (16-26 nm) (70-100 nm)

Adeno-associated viruses (AAV) : 

Adeno-associated viruses (AAV) -- do not stimulate inflammation in the host -- do not elicit antibodies against itself -- can enter non-dividing cells -- integrate successfully into sites fragile chromosomal sites (provide stable expression of the transgene) Can be ideal vectors as: Diseases treated with AAV vectors: 1) Coagulation factors IIIV and IX in hemophilia A and B 2) Regulated expression of insulin in diabetes

Bleeding Disorders: 

Bleeding Disorders von Willebrand disease (the most common) hemophilia A for factor 8 deficiency hemophilia B for factor 9 deficiency. hemophilia C for factor 11 deficiency A deficiency of a clotting factor can lead to uncontrolled bleeding. -- not enough of the factor OR -- mutant version of the factor Part of the clotting cascade

Hemophilia in European royal families: 

Hemophilia in European royal families

Untreated hemophilia: 

Untreated hemophilia www.dental.mu.edu/.../ www.pathguy.com; medgen.genetics.utah.edu

Hemophilia in human populations: 

Hemophilia in human populations Affects 1: 5,000 males 80% hemophilia A due to Factor VIII deficiency 20% hemophilia B due to Factor IX deficiency Results in spontaneous bleeding, which can be fatal Treated with prophylactic or therapeutic infusion of the deficient protein Correction to 1% of normal activity would reduce spontaneous bleeding; Correction to 10% of normal activity would eliminate most spontaneous bleeding;

Hemophilia A and B treatments: 

Hemophilia A and B treatments Extraction of a factors 8 and 9 from donated blood (>1000 donors), than purification. Injections of this material stops bleeding in hemophiliacs. Drawback: AIDS, hepatitis C. 90% of hemofiliacs in 90s were HIV+ 2) recombinant factor 8 and recombinant factor 9 made by genetic engineering are now available, very expensive 3) LIVER TRANSPLANTS

The changing prognosis of classic hemophilia due to HIV transmission: 

The changing prognosis of classic hemophilia due to HIV transmission Jones and Ratnoff, 1991. improvement in survival from 1971-1980 (corresponding to widespread treatment with lyophilized concentrates of Factor VIII)

Effect of AAV-mediated Hepatic Gene Therapy in Hemophilia B Dogs: 

Effect of AAV-mediated Hepatic Gene Therapy in Hemophilia B Dogs Normal: blood clots in about 8 to 10 minutes Diseased: blood clots in about 50 to 60 minutes

Injection of the vector into neonatal hemophiliac dogs: 

Injection of the vector into neonatal hemophiliac dogs 1-hour procedure of GT infusion, 15 month of stable expression, Blood clots in 20 minutes http://www.path.queensu.ca/present/lillicrap/7

Effect of AAV-mediated Hepatic Gene Therapy in Hemophilia B Dogs : 

Dr. Kenneth Brinkhous (North Carolina Univ) Effect of AAV-mediated Hepatic Gene Therapy in Hemophilia B Dogs Inject 1x1012 vector particles per kg into the portal vein of Hemophilia B dogs

Gene Therapy Approaches for Hemophilia:: 

Gene Therapy Approaches for Hemophilia: Liver-directed Muscle-directed Bone marrow-directed Transplantation of genetically-modified fibroblasts It does not really matter what tissue will produce the deficient protein as it will be secreted to the blood anyway

Ex Vivo vs. In Vivo Transfer into Liver : 

Ex Vivo vs. In Vivo Transfer into Liver Ex Vivo: Remove hepatocytes Modify cells in culture Reinject back to patient In Vivo: Inject vector into bloodstream

Clinical Trials in Patients:: 

Clinical Trials in Patients: Intra-Muscular injection of AAV for hemophilia B Intra-Venous injection of RetroV for hemophilia A Implantation of genetically-modified fibroblasts for hemophilia A Hepatic artery injection of AAV for hemophilia A

Intra-Muscular injection of AAV vector for hemophilia B: 

Intra-Muscular injection of AAV vector for hemophilia B Kay, High, Nature Genetics 24:257, 2000 Clot time Clot time F IX conc F IX conc

Intra-Venous injection of RetroV for hemophilia A: 

Intra-Venous injection of RetroV for hemophilia A Injected RetroV IntraVenous without a stimulus for hepatocyte replication (retroviruses can not infect non-dividing cells) Activity was less that 1% of normal Trial has been stopped No adverse effects were noted

Transplantation of genetically-modified Fibroblasts: 

Transplantation of genetically-modified Fibroblasts From the lecture of Prof. Ponder

Hepatic Artery Injection of AAV Vector for Hemophilia B (ongoing): 

Hepatic Artery Injection of AAV Vector for Hemophilia B (ongoing) Only 1/10 of dose injected in dogs were injected into the hepatic artery of patients; No evidence of expression to date; AAV was noted in semen for several months; Trial recently resumed with a medium dose of virus, but there are concerns about germline transmission

Conclusions:: 

Conclusions: No gene therapy approaches have evidence of strong long-term efficacy in patients up to date IM injection of AAV is too inefficient; Implantation of fibroblasts is very laborious/expensive and not very effective; Liver delivery of AAV or RetroV should work, but is not working yet…..

Curing Insulin-Dependent Diabetes Mellitus (IDDM) in mice and rats: 

Mice with inherited diabetes Rats after chemical destruction of their insulin-secreting beta cells Animals gained control over their blood sugar level and kept this control for over 8 months. Constructs injected into hepatic portal vein Curing Insulin-Dependent Diabetes Mellitus (IDDM) in mice and rats

CANCER GENE THERAPY: 

CANCER GENE THERAPY

Why cancer gene therapy? : 

Why cancer gene therapy? Balanced side effects and therapeutic cell killing Traditional chemotherapy lacks specificity:

CANCER TREATMENT: 

CANCER TREATMENT IDEAL GOAL: Completely eradicate tumor ATTAINABLE GOAL: Remove primary tumor (surgically) or achieve tumor shrinkage (chemotherapy) GOALS: Pain relief Infection combat (especially important for leukemias and lymphomas) Chemo-related problems combat

RADICAL STRATEGIES of the TUMOR TREATMENT: 

RADICAL STRATEGIES of the TUMOR TREATMENT

SURGICAL REMOVAL : 

SURGICAL REMOVAL MINUSES: Not able to catch and remove micrometastases; Not able to deal with ascytic (liquid) tumors

CHEMOTHERAPY: 

CHEMOTHERAPY CHEMOTHERAPY is the treatment of cancer with drugs that can destroy cancer cells. Ideal anticancer drug should be able to kill tumor cell and be harmless for any normal cell Problem: No clear differences between normal and tumor cells

FIRST PROBLEM WITH CHEMO = side effects: 

FIRST PROBLEM WITH CHEMO = side effects Normal cells with fast pace of divisions are also very susceptible to chemo (Cytoreduction)

SIDE EFFECTS of Chemotherapy: 

SIDE EFFECTS of Chemotherapy

SECOND PROBLEM WITH CHEMO = tumor resistance: 

SECOND PROBLEM WITH CHEMO = tumor resistance After rounds of chemotherapy and successful shrinkage of tumor and/or remission tumor cells become resistant to treatment

Slide30: 

From Prof. J. Koeller lecture (Univ of Texas)

CANCER GENE THERAPY: 

CANCER GENE THERAPY And other experimental cancer therapies (113 trials currently open in US in immunotherapy of cancer) 54% of immunotherapy trials dedicated to melanoma Delivery of the tumour-suppressor gene TP53 accounts for the next largest group Suicide Gene Therapies

Cancer Gene Therapy: Tumor Types: 

Cancer Gene Therapy: Tumor Types melanoma prostate ovar other http://www.path.queensu.ca/present/lillicrap/7

Slide33: 

Naked DNA and liposomes

Strategies for Cancer Gene Therapy: 

Strategies for Cancer Gene Therapy Directed to Tumor Directed to Host

Slide35: 

Strategies for Cancer Gene Therapy Directed to the Tumor Tumor suppressor delivery deliver human genes suppressing tumor growth Induction of apoptosis Deliver human genes that enhance apoptosis: ie. increase BAX Inhibit pro-apoptotic BCL-2 Working by the same principle as described for other gene therapies

Slide36: 

Strategies for Cancer Gene Therapy Directed to the Tumor Sensitizers to radiation and chemotherapy Deliver the gene that makes tumor cell more sensitive to DNA damage Pro-drug therapies (Suicidal therapies) Best examples: Thymidine Kinase that converts pro-drug Ganciclovir Cytosine Deaminase that converts prodrug 5-FC Directed to the Tumor

Slide37: 

Strategies for Cancer Gene Therapy Directed to the Host Anti-angiogenic strategies >40 natural anti-angiogenic molecules eg. Endostatin, angiostatin; suppress blood vessel support / cut oxygen out of tumor cells Confer Drug Resistance to Normal Host Cells improve chemoresistance in Hematopoietic progenitors

1. Tumor suppressor gene delivery (p53): 

1. Tumor suppressor gene delivery (p53) Introduction of p53 usually damaged in cancer tissue http://www.bol.ucla.edu/~kcrazy/Oliff51.gif Tumor cell Normal cell

2. Genetic pro-drug activation therapy (GPAT): 

Normal breast cells never express ERBB2 (therefore, they will never express GT gene under ERBB2 promoter) Suicide gene converts harmless 5-FC pro-drug to the cytotoxic 5-FU drug Death = Only for tumor cells. Tumor-specific Suicide promotor + drug activating gene 2. Genetic pro-drug activation therapy (GPAT) GT construct: Cytosine Deaminase gene under ERBB2 promotor. Suicide http://www.sghms.ac.uk/depts/ogem

HSV-TK based GPAT strategy: 

The HSV-tk/Ganciclovir combination is now in multicenter, phase III clinical trials in glioblastoma patients HSV-TK based GPAT strategy stable expression of HSV-timidine kinase could increase cell sensitivity to GCV by up to 2000-fold

Examples of suicide schemes: 

Examples of suicide schemes Linamarase = beta-glucosidase, to convert the cyanogenic glucoside substrate, linamarin, into glucose and cyanide. From cassava Production of the cyanide ion that diffuses freely across membranes. In culture 10% lis-positive glioma cells are sufficient to eliminate the entire glioma cell culture in 96 h.

Bystander effect: 

Bystander effect Herpes simplex virus (HSV-1) structural protein VP22 able to travel to neighboring cells; any vector that contain VP22 on its surface enhances bystander effect of the gene delivered

3. Make normal cells of the patient resistant to chemotherapy (body-directed cancer GT): 

3. Make normal cells of the patient resistant to chemotherapy (body-directed cancer GT) Blood vessel Cancer cells express MDR1 (multi-drug resistance protein) Cancer cells outplay hematopoietic cells in chemotherapy mass death game MDR1 actively pumps toxic hydrophobic compounds (drugs) across the plasma membrane making cancer cells resistant to drug

To INTRODUCE MDR1 gene into hematopoietic cells so they can play with cancer cells equally: 

To INTRODUCE MDR1 gene into hematopoietic cells so they can play with cancer cells equally Gene therapeutic IDEA It may be useful as protection of cancer patients from the myelosuppressive side effects of chemotherapy.

Ex vivo scheme for GT protection of hematopoietic cells against chemotherapy: 

Ex vivo scheme for GT protection of hematopoietic cells against chemotherapy Hematopoietic cells taken from bone marrow CANCER Patient treated with CHEMO Hematopoietic cells transfected with MDR1 gene Hematopoietic cells Multiplied in vitro CHEMO-resistant cells are transferred back to patient

CHEMO-resistance genes used for ex vivo GT (body-directed): 

CHEMO-resistance genes used for ex vivo GT (body-directed) MDR1 pump (resistance to paclitaxel = Taxol and other drugs) Mutant DHFR enzyme (resistant to methothrexate drugs) O6-alkylguanine-DNA-alkyltransferase gene (Resistance to alkylating agents)

OTHER MODERN METHODSof tumor treatment: 

OTHER MODERN METHODS of tumor treatment 1. Immunotherapy (with antibodies) Monoclonal antibodies to growth factor receptor Monoclonal antibodies for directed tozin delivery Immunomodulation Cancer vaccines 2. siRNA and antisense gene suppression approaches 3. Oncolytic viruses