gene therapy 1 guo

• GENE THERAPY - involves transfer of genetic material into host cells to cure or favorably modify the clinical course of a disease. allows dr. to treat a disorder by inserting. gene into patients cells instead of drugs or surgery. replacing mutated w healthy gene
• GENE THERAPY APPLICATIONS

  • replacement of defective genes such as CFTR gene in cystic fibrosis
  • supplying a gene product in large quantities in an unregulated manner such as factor IX in hemophilia
  • introducing genes into a cell that dont normally express it as immunopotentiaion genes in cancer cells
  • Introducing protective genes into target cells such as viral- specific ribozymes to treat HIV-1 infections • Modulation of immune response such as tolerance induction in autoimmune diseases • DNA vaccinations such as West Nile virus DNA vaccine for veterinary applications
• REQUIREMENTS FOR SUCCESSFUL GENE THERAPY PRODUCTS

  • selecting the right gene - understand the disease
  • identifying and accessing target cells that require the treatment
  • appropriate gene delivery system
  • proof of principle, safety, efficacy
  • suitable manufacturing and analytical processes
• TARGET CELLS

  • SOMATIC gene therapy
  • not inhertible
  • where dos it only occur ? in target cells
  • aim to cure disease only in PATIENT, not their descendants
  • GERMLINE GENE THERAPY
  • inheritable
  • will pass onto next generation
  • genetic modification of germ cells
• ADVANTAGES OF TARGETING LYMPHOCYTES FOR SOMATIC CELL GENE THERAPY

  1. Relatively long-lived
  2. Readily obtainable from peripheral blood
  3. Easy to manipulate for gene transfer
  4. No inactivation of gene expression during differentiation
  5. Can be depleted post-transfer, a useful safety back-up
  6. Can be used to supply blood borne gene products: able to secrete large amounts of protein: Hemophilia B (factor IX)
  7. Potentially useful for manipulating immune responses: Car T cell immunotherapy
• EX VIVO 

  • PATIENT X CELLS - gene transfer - select cells -amplify - return cells back into patient genetically - OUTSIDE THE BODY 

  Advantage: • Does not require tissue specific vectors • Very high transfer efficiency • Target cells can be manipulated / amplified

  Disadvantage: • Can be used for limited target cells such as blood cells • Cells need to retain the ability to “home” and function normally post-transfer • Invitroartifacts
• IN VIVO - gene transfer into target cells occurs in patient body .. INSIDE THE BODY 

  Advantage: • Can target all body tissues • No in vitro artifacts

  Disadvantage: • Specificity of gene transfer can be an issue • Less invasive
• When used to transfer genes into target cells, the viruses are called viral vectors
• HARMLESS VIRUSES

  • REMOVE VIRUS GENES, REPLACE WITH GENE U WANT
  • PACK THIS GENE INTO VIRAL PARTICLES TO sneak into target cells
  • viruses called viral vectors, which carry genes into the cell and integrate/insert the gene into host cell chromosomes (SPY)
• gene delivery systems : viral

  1. RETROVIRALand LENTIVIRAL 2) ADENOVIRAL 3) ADENO-ASSOCIATED VIRAL 4) HEPES SIMPLEX VIRAL
• gene delivery systems : nonviral

  1. LIPOSOMES 2) NANOPARTICLES 3) BIOLISTIC GENE GUN 4) NAKED DNA
• RETROVIRUSES USED FOR GENE DELIVERY

  • single stranded positive-sense RNA viruses
  • a type of RNA virus that inserts a copy of its genome into the DNA of a host cell that it invades, changing the genome of that cell
  • infection will persist indefinitely
• How does there right RNA get packaged into virion particle ?

  • RNA —> dsDNA —> RNA —> polypeptide
• • POL
  • viral enzymes
  • for viral infection
  • ex. reverse transcriptase, integrase, protease
• • ENV
  • viral envelope (indicates tropism)
  • for recognizing specific receptors on host cell, initiating the infection process
  • ex. transmembrane subunit, surface subunit
• • GAG
  • viral core structural proteins
  • for viral core particle
  • ex. matrix, nucleocapsid, capsid
• retrovirus life cycle

  1. attachment
  2. entrance
  3. reverse transcription
  4. integration
  5. transcription and processing
  6. translation and processing
  7. assembly
  8. budding
• • cis
  • DIRECTLY ACTIVE AS NUCLEIC ACIDS

  1. 5' long terminal repeat
  2. primer binding site (PBS) for first strand DNA synthesis during reverse transcription
  3. psi directs packaging of the genomic RNA into the virion
  4. polypurine tract - primer binding site for second strand DNA synthesis during reverse transcription
  5. 3' LTR in the DNA form acts as a polyadenylation signal, RNA (genomic) form contains sequences important for reverse transcription processes
• • trans
  • PROTEIN CODING SEQUENCES
• what happens when the packaging signal psi is deleted from the viral genome?

  • psi directs packaging of genomic rna into the virion
  • since this works with packaging, if there is malfunction, there is no packaging aka would lead to EMPTY VIRUS PARTICLES AND NO VIRAL DNA
• Recombination between helper and vector virus could regenerate : Replication Competent Virus
• Replication defective retroviral vector systems are made by separating the cis and trans genetic functions of the virusinto a vector construct, which contains the cis sequences, and helper or packaging plasmids, that encode the viral proteins
• transcription of 2nd gene is less efficient
• Viral vectors have natural host cell populations that they infect most efficiently, which limits natural host cell ranges.
• pseudo typed viruses

  : the envelope proteins consist of the parts of the viral protein necessary for incorporation into virion and the sequences meant to interact with specific host cell proteins
• Simplest retroviral vector

  • retrovirus in which all the trans sequences were replaced only by the gene of choice
  • main limitation : can only express one gene
• advantages of using retroviral vectors

  • efficient and stable integration
  • controllable host range via envelope pseudo typing
  • capable of delivering upto 8 kbp of exogenous sequences

  disadvantages of using retroviral vectors

  • can infect only dividing cells
  • difficult to obtain high titers / concentration
  • 8kbp might not be enough

  safety concerns for using retroviral vectors

  1. production of replication-competent virus
  2. insertional mutagenesis
• LENTIVIRAL VECTORS

  • more complex type of retroviruses
  • can infect both dividing and non dividing cells
  • integrate permanently into host genome
  • human lentiviruses that have been explored for gene delivery systems are HIV-1 and HIV-2

  alternatives

  • SIV
  • FIV, EIAV
  • HIV
• adenoviral vector delivery system

  • NON ENVELOPED DOUBLE STRANDED DNA VIRUS
  • mild respiratory infection and other illnesses
  • therapeutic gene extra chromosomal dna DOES NOT INTEGRATE
• adenoviral vectors

  • high titers
  • ability to infect a broad range of non-dividing and dividing cells
• adenoviruses are strongly immunogenic

  • destroy cells expressing viral proteins

  Limitation

  1. pre-existing immunity
  2. leaky expression of adenovirus proteins from genes that were not deleted

  improved adenoviral vectors

  • limited number of repeated treatment with adenoviral vector
• adenovirus vectors

  main advantages

  • Large capacity of transgene (36kb)
  • High titers
  • Ability to infect a broad range of cells
  • Infect both dividing and non-dividing
  • No of evidence for chromosomal integration
  • Viral genome stable and does not undergo rearrangement at a high rate
  • Low pathogenicity of virus in humans

  main disadvantages

  • very immunogenic
  • do not integrate into hosts genome
  • transient expression

  could be good for …

  • modulate immune response
  • develop vaccines
  • cancer treatment
• • adeno-associated virus (AAV) is a non pathogenic, defective virus that requires a helper virus to supply the machinery for producing infectious particles
  • packaging size of the expression cassette that will be placed between the two ITRs
• Having temporally modulatable expression

  • Use to drive gene expression . Muscle-derived but not centrally derived transgene GDNF is neuroprotective in G93A-SOD1 mouse model of ALS, 2006, Exp. Neurol

  • Can target the viral vector to specific cells using a designer Cap protein

  • Can use bidirectional promoters to drive multiple gene expression
• Adeno-Associated Virus (AAV) Vectors Main Advantages:

  • Small, easy to manipulate virus
  • Infect a wide range of both non-dividing and dividing cells
  • Low immunogenicity
  • Not associated with any known human disease

  Main Disadvantages:

  • Limited packaging capacity (~5kb)
  • Requires adenovirus as helper virus (although producer cell lines have recently been developed)
• Herpes Simplex type I Virus (HSV) -

  • A double-stranded DNA virus • Neurotropic Virus – tissue specific gene transduction

  • Treat CNS (central nervous system) diseases such as Parkinson’s Disease

  • Can be selectively depleted by treating with Ganciclovir (virus naturally encodes HSV thymidine kinase (HSV-TK) gene
• viral gene delivery systems

  1. retroviral vectors
  2. lentiviral vectors
  3. adenoviral vectors
  4. adenoassociated viral vectors
  5. herpes simplex type 1 virus
• envelope virus - lentivirus
• nonenvelope virus - adeno associated viruses. have a protein based capsid as outer shell
• Gene augmentation


  Treat loss-of-function defect by adding functional gene