LATEST ADVANCES IN MEDICAL GENOMICS PART 1 Dr

LATEST ADVANCES IN MEDICAL GENOMICS (PART 1) Dr. Kumitaa Theva Das TMR 501 19 November 2014

TOPICS COVERED - Conventional methods - Latest therapeutic techniques - Delivery methods - Genome sequencing - Designer humans - Implications of genome editing - Hurdles and risks - Ethical issues

GOALS - To know conventional methods - To understand latest techniques including aptamers, RNAi and CRISPR - To understand delivery mechanisms, particularly lentiviral vectors - To know essential requirements, benefits, risks and problems associated with gene therapy - To be able to debate ethical issues related to gene therapy

DEFINITION - What is genetics? - The study of heredity and the variation of inherited characteristics. - What is genomics? An area within genetics that concerns the sequencing and analysis of the function and structure of the genome (the complete set of DNA within a single cell of an organism). - What is medical genomics? - Medical genomics is the specialty of medicine that involves the diagnosis and management of hereditary disorders found in the genome.

Latest advances in medical genomics

CONVENTIONAL APPROACHES - Most genetic disorders cannot be cured - Primarily because there is little understanding of the basic metabolic or molecular defect - If understood, treatment can be done by metabolic manipulation, such as: A) Dietary restriction B) Replacement C) Inhibition D) Supplementation

DIETARY RESTRICTION - Usually involves amino acid catabolic pathways. - Example: Diet restricted in phenylalanine circumvents neurological damage in PKU (phenylketonuria). - Results of treatment best as soon as diagnosis is made.

REPLACEMENT - Most successfully treats singlegene defects. - Example: Congenital hypothyroidism, which results from defects in the thyroid gland or its major product, thyroxine. - In some countries, neonatal screening is conducted and thyroxine is administrated.

INHIBITION - Can be used to modify inborn errors. - Example: Familial hypercholesteromia, where cholesterol synthesis is upregulated. - Statin inhibits 3 -hydroxyl-3 methylglutaryl coenzyme A reductase, the rate limiting enzyme of cholesterol synthesis.

SUPPLEMENTATION - Typically for deficient vitamin or co-enzyme. - Example: Scurvy, characterized by weakness, bruising, bleeding gums, is caused by a Vitamin C deficiency. - Can be corrected with vitamin supplementation.

MOLECULAR TREATMENT - Treatment done based on understanding of molecular mechanisms. - Typically done when there is a mutant protein. - Usually enables folding of protein, or increment of protein to restore biochemical homeostasis. - For mutant protein, treatment includes: A) Enhancement of mutant protein function B) Protein augmentation

ENHANCEMENT OF PROTEIN FUNCTION - Usually done with small molecule therapy. - Allows for mutant polypeptide to fold normally. Example: Curcumin inhibits binding of F 508 in mice (prevents cystic fibrosis).

PROTEIN AUGMENTATION - Well established, safe and effective. - Involves protein in plasma or extracellular fluid. - Example: Prevention of bleeding seen in hemophilia with factor VIII.

LATEST ADVANCES IN MEDICAL GENOMICS - Advances involve modification of the genome. - Includes: A) RNA based agents B) Protein based agents C) Gene therapy D) Vectors

RNA BASED AGENTS - RNA based agents include: A) Ribozymes B) Antisense C) Aptamers D) RNAi

RIBOZYMES - RNA molecule - Catalyzes specific biomolecule reactions (like enzymes) - Often have hairpin/hammerhead structure that allows cleavage of specific site - Currently a ribozyme against HIV RNA, cancer and amyotrophic lateral sclerosis is in clinical testing

ANTISENSE - Nucleic acid that is complementary to m. RNA - Binding inactivates gene - About 20 in advanced clinical trials including for Ebola, cancer and HIV - Two approved include cytomegalovirus retinitis and familial hypercholesterolemia

APTAMERS - Can be oligonucleotide or peptide - Has advantages over antibody as it can be: A) Engineered in a test tube B) Produced by chemical synthesis C) Desirable storage properties D) Elicit no immunogenicity - Have been designed to VEGF, dopamine, oncogene - Approved for treatment against age-related macular degeneration

RNA INTERFERENCE - RNAi can be used to degrade RNA - si. RNA and mi. RNA can bind to m. RNA - Found in eukaryotes - Initiated by Dicer which cleaves long ds. RNA into short oligonucleotides - Short oligonucleotides separated into ss. RNA: A) Passenger strand B) Guide strand

RNA INTERFERENCE - Guide strand incorporated into RISC and binds to complementary m. RNA - Complex induces cleavage by Argonaute (catalytic component) - Have been shown to work against cancer, herpes, hepatitis A and B, HIV - In clinical trials against macular degeneration

PROTEIN BASED AGENTS - Protein based agents include A) Fusion inhibitors B) Zinc fingers C) TAL D) CRISPR/Cas 9

FUSION INHIBITORS - Also known as entry inhibitor - Interferes with binding, fusion and entry of virus into the host cell - Prevents virus replication - Among approved inhibitors include Maraviroc which binds to CCR 5 and inhibits HIV binding

ZINC FINGERS CHIMERA - Composed of zinc finger proteins (DNA binding domain) with an additional domain such as transcriptional activator, repressor, methylation domain or nuclease - Have been used to target various genes in Drosophila melanogaster, C. elegans, rats, zebrafish and humans - Zinc finger nuclease that targets CCR 5 (co-receptor for HIV) is currently in clinical trials

TAL EFFECTOR - Transcription activator-like effector domain which is similar in function to zinc fingers - Have also worked in targeting genes in plants, zebrafish, rats and human - However no successful delivery method as of today

CRISPR/CAS 9 - Prokaryotic immune system (adaptive immunity) - Analogous to RNAi in eukaryotes - Composed of guide RNA (cr. RNA and tracr. RNA) and Cas 9 (nuclease)

CRISPR/CAS 9 - Similar in function and application to zinc fingers: A) Activator B) Repressor C) Nuclease D) Marker E) Diagnostic tool

DELIVERY VECTORS IN GENE THERAPY - A) Viral - B) Non-viral

VIRAL VECTORS - Lentivirus - Adeno-associated virus

LENTIVIRUS - Simple RNA virus with structural genes removed - Engineered to render them unable to replicate - Non-toxic - Stably integrated - Large enough to accommodate gene of interest - Works in all cell types - Works in non-dividing cells as well

ADENOVIRUS - Can obtain high titers - Capable of infecting a wide variety of cells - Can infect dividing and non-dividing cells - Can accommodate large inserts (30 -35 kb) - But have caused death in gene therapy trial - Have also been known to elicit strong immune response

ADENO-ASSOCIATED VIRUS - Have no adverse effects on humans - Most exist episomally - However a disadvantage is that they can only accommodate small inserts

NON-VIRAL VECTORS - Naked DNA (c. DNA with regulatory elements) - Liposomes - Protein-DNA conjugate (DNA complexed to peptide) - Artificial chromosomes (parts of chromosome combined with c. DNA)

TYPES OF GENE THERAPY - A) Somatic gene therapy - B) Germline gene therapy

SOMATIC GENE THERAPY - Therapeutic genes are transferred to somatic cells - Modification will not be inherited by offspring - Some therapies are in clinical trials including for hemophilia, thalassemia, and cystic fibrosis

GERMLINE GENE THERAPY - Germ cells (sperm or eggs) are modified by introduction of genes, integrated into the genome - Germ cells will form an embryo, continue to divide until all cells carry modification - Inherited by offspring - Effective for hereditary diseases - Higher risk than somatic gene therapy

LATEST ADVANCES IN MEDICAL GENOMICS (PART 2) Dr. Kumitaa Theva Das TMR 501 27 November 2014

REVIEW 1. What is the central dogma of biology? DNA RNA Protein

REVIEW 2. What do you call the process from DNA to RNA and RNA to protein? DNA RNA Transcriptio n Translation Protein

REVIEW 3. What are the protein based gene therapy agents and what are the RNA based gene therapy agents? DNA RNA Transcriptio n Translation Protein Fusion inhibitor, zinc finger, TAL, CRISPR proteins Ribozymes, antisense, aptamers. RNAi

REVIEW 4. Out of all the gene therapies, which two are analogous to each other? DNA Transcriptio RNA n Translation Protein Fusion inhibitor, zinc finger, TAL, CRISPR proteins Ribozymes, antisense, aptamers. RNAi

REVIEW 5. What are 3 key components of CRISPR and RNAi? DNA Transcriptio RNA n Fusion inhibitor, zinc finger, TAL, CRISPR proteins Ribozymes, antisense, aptamers. RNAi Translation Protein cr. RNA, tracr. RN A, Cas 9 Dicer, RISC, Argonaut e

REVIEW 6. What are some of the modifications that can be done to the genome? Agent + activator domain = Gene activation = Gene overexpression

REVIEW 6. What are some of the modifications that can be done to the genome? Agent + repressor domain = Gene silencing = Gene knockdown

REVIEW 6. What are some of the modifications that can be done to the genome? Agent + marker = Diagnostic tool = High throughput screening

REVIEW 6. What are some of the modifications that can be done to the genome? Agent + cleavage domain = Gene knockout

REVIEW 6. What are some of the modifications that can be done to the genome? Agent + cleavage domain + donor DNA = Gene replacement

REVIEW 7. List 3 types of viral vectors that can be used to deliver the RNA and the protein based agents. DNA RNA Transcriptio n Translation Protein Fusion inhibitor, zinc finger, TAL, CRISPR proteins Ribozymes, antisense, aptamers. RNAi Lentivirus, adenoassociated virus

REVIEW 8. List some of the features found in the viral vector that makes it a suitable delivery vector. DNA RNA Transcriptio n Translation Protein Fusion inhibitor, zinc finger, TAL, CRISPR proteins Ribozymes, antisense, aptamers. RNAi Lentivirus, adenoassociated virus Can target all cell types, nondiving cells, stable, large

END OF REVIEW FOR PART 1

SPECULATIVE USES FOR GENE THERAPY IN GENOMICS - Personalized medicine - Genetic counseling - Human genetic engineering

WHOLE GENOME SEQUENCING Year Cost 2007 $ 1 million End of 2007 $ 350, 000 2010 $ 50, 000 2014 $ 1, 000

PERSONALIZED MEDICINE - Healthcare that uses molecular analysis with medical decisions tailored to individual patients - Might increase responsiveness to drugs - Might allow for earlier detection, diagnosis and intervention - Might allow for genetic disorders to be corrected

Double

GENETIC COUNSELING - Process in which patients at risk of an inherited disorder are advised on consequences of disorder and management options - Results from test may reveal: A) Downs syndrome B) Sickle-cell anemia C) Muscular dystrophy D) Mental retardation

Alcohol tolerance

ESSENTIAL REQUIREMENTS FOR GENE THERAPY - Known identity of molecular defect - A functional copy of the gene - Knowledge of the mechanism - Favorable risk-to-benefit ratio - Appropriate regulatory components for the transferred gene - Appropriate target cell - Strong evidence of efficacy and safety - Regulatory approval

PROBLEMS WITH GENE THERAPY - Short lived nature of gene therapy - Unwanted immune response - Problems with viral vectors: Possible toxicity - Difficult to treat multi-gene disorders - Insertional mutagenesis (might induce tumor) - Cost is extremely high - Possible death

QUESTI ONS?