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Flashcards in Viruses as Therapeutics Deck (24):
1

Three uses of viruses in therapeutics

  1. Provide Gene Replacement Therapy
  2. Create recombinant vaccines to promote immunity to infectious agents and tumors
  3. Act as targeted killers of cancer cells

2

Gene therapy definition

The transfer of new genetic material to the cells of an individual with resulting benefit to the individual

3

Germline gene therapy vs. Somatic gene therapy

Germline: modifies gene that may be passed on to subsequent generation

Somatic: genetic modifications restricted to somatic cells, with no effect on the germline

4

Two general approaches to delivery

  • ex vivo: cells removed from patient and exposed to the vector in cell culture
  • in vivo: vector introduced directly into patient

5

Top 3 indications addressed by gene therapy clinical trials

  1. Cancer diseases
  2. Monogenic disease
  3. Infectious diseases

6

Top 3 gene types transferred in gene therapy clinical trials

  1. Antigen
  2. Cytokine
  3. Tumor supressor

7

Gene delivery agents

  • Viruses
  • Chemical - transfection
  • DNA loaded vesicles (liposomes)
  • DNA-protein complexes
  • Physical (microinjection - direct injection of DNA)

8

X-SCID is due to molecular defects in the gene for the _______ ______ ______

common gamma chain (γc)

9

Retrovirus vectors for gene delivery

  • Replace viral genes with exogenous genes
  • Retain LTRs (integration), Ψ sequence (packaging)
  • Supply deleted gene products in trans
    • Packaging cell lines
    • Helper virus, helper plasmids
  • Infect cells to introduce exogenous genes

10

Creation of packaging line

  • Start with normal cultured cells
  • Transfect plasmid DNAs that will express gag, pol, and env proteins in cells
  • Genes encoding gag, pol, and env will integrate into the genome of the packaging cells
  • gag, pol and env proteins will be constitutively expressed in the packaging cells

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11

Transfecting vector plasmid with gene of interest into packaging cell line to produce vector particles

  • Vector plasmid will integrate into the genome of the packaging cells
  • Vector RNA will be transcribed by host-cell RNA polymerase
  • Vector RNA will be packaged into vector particles by the viral proteins that are being synthesized in the producer cells

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12

Retrovirus vectors

Advantages:

Disadvantages:

Advantages:

  • Efficient delivery of foreign genes
  • Control over host range, cell and tissue tropism

Disadvantages:

  • Random integration into chromosomes, insertional mutagenesis
  • Maintaining high levels of gene expression
  • Dependence on target cell proliferation with MMLV (leukemia)-based vectors

13

Retrovirus LTR contains a stron T-cell specific _______

enhancer

14

Lentivirus Vectors

Preintegration complex with Vpr, integrase, and matrix protein will cross the nuclear membrane in nondividing cells

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15

Lentivirus Vectors - Safety Concerns

  • Multi-plasmid systems for creating packaging cells
  • Self-inactivating vectors
    • Deletion in U3 region of right LTR
  • Control of vector tropism

16

Adenovirus - Basic Biology

  • Linear dsDNA genome
  • Replicates in the nucleus - no integration
  • Gene transcription regulated with immediate early, early and late classes of mRNAs

17

Creating adenovirus construct

  • Delete certain viral genes to make room for therapeutic gene of interest
  • Some deleted viral genes are essential for the replication of adenovirus so they must be provided in trans by the complementing cell line

18

Deliver of adenovirus vector construct

  • Transfect vector DNA construct into the complementing cell line (293 cells) to produce vector particles
  • Vector particles are used to deliver the therapeutic gene of interest into the target cells of the patient
    • Vector DNA enters the nucleus of the target cell but DOES NOT integrate
    • Therapeutic product synthesized in the cells that have been infected

19

Adenovirus Vectors - Problems

  • Short term gene expression - no integration of recombinant DNA into host genome
  • Immune response - limits success of repeated use of vectors
  • Size restrictions on inserted genes
  • Solutions: Better packaging cell lines - "gutless vectors"

20

Vaccinia Virus Expression Vectors - Biology

  • Large, dsDNA genome
  • Flexibility in the size of DNA that can be packaged
  • Cytoplasmic replication
  • Virus encoded enzyme
    • RNA polymerase
    • capping, methylating, polyadenylating
  • Viral promoters
    • Viral RNA polymerase, not cellular pol II

21

Basic strategy  for foreign gene expression with vaccinia vectors

  • Plasmid construction
    • Foreign gene + vaccinia promoter
  • Chimeric gene → vaccinia genome (homologous recombination)
  • Foreign gene → non-essential site in vaccinia genome
    • TK (Thymidine kinase) gene is the most popular site
    • Selection: TK(-) virus, TK(+) virus-BUdR sensitive

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22

Viruses as Oncolytics

Oncolytic Wild Viruses - some wild-type viruses have natural oncolytic activity in human tumors

Genetically manipulated viruses specifically targeted to infect and kill cancer cells

23

Conditionally replicative Adenovirus (CRAd)

Modifications in adenovirus that allow tumor-specific tropism:

  • Deletion of E1A or E1B genomic regions - viruses will only replicated in cells with specific dysfunctions in cell cycle checkpoint pathways, like some cancer cells
  • Incorporation of tumor-specific promoters
  • Improvement of transduction efficiency in tumor cells, often accomplished by altering the virus cell attachment protein

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24

Reovirus (wild-type strain)

No modifications by recombinant DNA

  • Inherent tumor selectivity for cells with an activated Ras activity
  • In normal cells reovirus replication is restricted by the activation of the RNA-activated protein kinase (PKR)