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Immunology 3 > #17 Virus as therapeutics > Flashcards

#17 Virus as therapeutics Flashcards

1
Q

A virus can:

A

gene replacement therapy
made recombinant vaccines (immunity against tumors and infections)
act as targted killers of cancer cells

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2
Q

transfer of new genetic material to cells of ind w/ benefical results

A

Gene therapy

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3
Q

modifies gene that may be passed onto subsequent generations

A

Germline gene therapy

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4
Q

genetic mods restricted to somatic cells, NO effect on germline

A

Somatic gene therapy

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5
Q

Two delivery options for gene therapy
ex vivo:
in viv:

A

cells removed from pt and exposed to vector in cell culture

-vector introduced directly into pt

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6
Q

Example of in vivo gene therapy:

two methos

A

Replace deficient factor (like giving CF gene to pts with CF)
Expression of foreing gene (would be beneficial to infect brain tumor with a virus so that when we give meds to tx virus, tx tumor)

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7
Q

Gene therapy trials are adressing:

A

lots of cancer disorders

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8
Q

Gene types transfered in gene therapy trials, main ones

A

see antigen, cytokine, tumor suppresor and suiciced gene transfer

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9
Q

Gene delivery agents

A

Viruses!!! they are great at delivery

-also see chemicals/DNA loaded vesicles/ DNA protein complexes

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10
Q

What viruses are being used for gene therapy trials

A

Adenovirus, Retrovirus, Poxvirus~ we’ve talked of these

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11
Q

retrovirus gene therapy history:

terminal melonma

A

given lymphocytes marked with ‘neo’ gene via a retrovirus vector

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12
Q

Ex of Ashanti with T cell deficiency

A

Tcells genetically corrected, then returned to her and now 20–25% of all her T cells ahve the gene the retrovirus introduced

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13
Q

SCID story

A

used retroviral gene transver to CD34+ cells

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14
Q

X-linked severe combined immunodeficiency
• Patients present with no T or NK lymphocytes and non- functional B cells
• If untreated, X-SCID is fatal in the first few years of life due to overwhelming susceptibility to infections
• X-SCID is due to molecular defects in the gene for the

A

common gamma chain (γc)

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15
Q

Fixing SCID

kids with no T/NK and nonfnx B cells

A

Replace viral genes with exogenous genes

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16
Q

During Gene Delivery for SCID:
Retain LTRs (_______,) psi sequence (_______) Supply deleted gene products in trans
– packaging cell lines
– helper virus, helper plasmids
Infect cells to introduce exogenous genes

A

integration

for packaging

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17
Q

Basically, you delete the gag/pol/env and put in what you need, but we keep the LTR region which includes

A

U3-R-U5

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18
Q

With our modified product from the retrovirus, we do all the same steps up to ________ and _________ but NO virions made, just the proteins of interest

A

circulization and integration

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19
Q

We start with a culture cell then transfect with:

A

gag, pol and env

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20
Q

Creation of packaging cell line
start with normal cultured cells and
transfect plasmid DNAs that will express

A

gag, pol, and env proteins in the cells

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21
Q

Creating packaging cell line:(post transfection)
genes encoding gag, pol, and env will _______ into the genome of the packaging cells and will be constitutively expressed in the packaging cells

A

integrate

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22
Q

Transfect vector plasmid with gene of interest into the packaging cell line to produce

A

vector particles

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23
Q

Vector plasmid will integrate into the genome of the packaging cells vector
RNA will be transcribed by __________

A

host-cell RNA polymerase

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24
Q

_________will be packaged into vector particles by the viral proteins that are being synthesized in the producer cells

A

vector RNA

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25
Q

Transfect vector plasmid with gene of interest into the packaging cell line to produce vector particles
…….

A

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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26
Q

The cells will die after time… but they have made

A

vector containing virus that can infect a target cell –> reverse transcriptase–> integrate gene

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27
Q

Advantages of Retrovirus vectors

A

– efficient delivery of foreign genes

– control over host range, cell and tissue tropism

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28
Q

Disadvantages of retrovirus vectors

A

– “random” integration into chromosomes, insertional mutagenesis
– maintaining high levels of gene expression
– dependence on target cell proliferation with MMLV-based vectors

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29
Q

____LTR contains strong, T cell specific enhancers

A

MLV
—-integrates in region
we can get over expression of U3 regions because our gene inserts a a MLV enhancer region?

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30
Q

Lentivirus Vectors:

preintegration complex with Vpr, integrase, and matrix protein will cross the nuclear membrane in _______

A

nondividing cells

31
Q

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

A

Lentivirus Vectors:

32
Q

Key parts of lentivirus

A

Vpr, integrase and matrix proteins

33
Q

Lentivirus Vectors Addressing Safety Concerns

A
  • Multi-plasmid systems for creating packaging cells
  • Self-inactivating vectors – Deletion in U3 region of right LTR
  • Control of vector tropism
34
Q

Lentivirus is pretty sweet bc it has this deleted in the right of the LTR

A

U3 region that causes ehnaced expression

35
Q

has multi-plasmid systems for creating packaging cells

A

lentivirus

36
Q

Basic Biology – linear, dsDNA genome – replicates in the nucleus, no integration
– gene transcription regulated with immediate early, early and late classes of mRNAs

A

Adenovirus Vectors

37
Q

Adenovirus Vectors
lindear dsDNA and replicates in the ______
does it have integration?

A

replicates in nucleus

NO NO integration so no insertional mutagensis

38
Q

What vector virus replicates in the nucleus and doesn’t integrate into host DNA

A

Adenovirus vector

39
Q

How is gene trascription regulated with adenovirus

A

early, early and late classes of mRNAS

40
Q

Create adenovirus vector construct

  1. delete certain viral genes to make room for therapeutic gene of interest
  2. some deleted viral genes are essential for the replication of adenovirus so they must be
A

provided in trans by the complementing cell line

41
Q

(adenovirus vecotr contstruct)

Transfect vector DNA construct into the complementing cell line (293 cells) to produce

A

vector particles

42
Q

Once we’ve made adenoviris vector particles, what happens

A

Vector particles deliver the therapeutic gene of interest into the target cells of the patient

43
Q

therapeutic product is synthesized in the cells that have been infected by the adenoviral vectors but the vector DNA does not

A

integrate

44
Q

(Adenovirus)
vector DNA enters the nucleus of the target cell but does not integrate
therapeutic product is synthesized by what?

A

the cells that have been infected by the adenoviral vectors

45
Q

Adenovirus Vectors - Problems
Short term gene expression:
Immune response
Size restrictions on inserted genes

A
  • -no integration of recombinant DNA into host cell genome

- limits success of repeated use of vectors and E3 region deltions will increase immune response

46
Q

Solutions to Adenovirus issues

A

better packaging cell lines, “gutless vectors”

– better understanding of immune response to adenovirus

47
Q

Vaccinia Virus Expression Vectors

• Related Biology –

A

large, dsDNA genome (187,000 bp) – flexibility in the size DNA that can be packaged
– cytoplasmic replication
– virus encoded enzymes
– viral promoters

48
Q

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 gene is the most popular site

A

TK gene is most popular because it’s non-essential

49
Q

How i believe this whole recombinant plasmid shit works

A

have plasmid DNA that we added our foregin gene/vaccinia DNA–>
transfected a cell with this + infectious vaccinia virus –> get recombination of vaccinia sequences–>
replication of recombiant vaccina DNA with foreign gene–> maturation –> release of recombinat vaccinia virus containing the foreign gene

50
Q

The Foreign gene thats recombinant of vaccinia virus is used to make vaccination with a live virus capable of:

A

producing foreign immunizing antiG

51
Q

Chimps that got the recombinant vaccine and were then infected with HBV

A

had a primed immune response and did great

52
Q

stable integration, long-term gene expression

potential for insertional mutagenesis replication incompetent

A

Retrovirus vectors:

53
Q

Key point

retrovirus have________ integration and______ gene expression

A

stable

long term

54
Q

these have potential insertional mutagensis

A

Retrovirus

55
Q

these are replication incompetent

A

retrovirs

56
Q

vector has no integration and short term gene expression

A

adenovirus

57
Q

this vector sucks because we see vigours immune response to the vector

A

adenovirus

58
Q

replication incompetent vectors

A

retro
adeno
pox

59
Q

_____ vectors: no integration

replication competent live, attenuated vaccines

A

Poxvirus (vaccinia)

60
Q

which vector makes live attenuated vaccines

A

poxvirus

61
Q

Canarypox is useful for

A

HIV vaccine because they dont complete replication in human cells and no lysis

62
Q
  • Myxomavirus
  • Bovine herpesvirus 4
  • Reovirus
  • Newcastle disease virus
  • Coxsackie virus
  • Vesicular stomatitis virus
  • Parvovirus
A

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

63
Q

Genetically manipulated viruses specifically targeted to infect and kill cancer cells:

A
  • Adenoviruses
  • Herpes simplex viruses
  • Vaccinia virus
64
Q

adenovirus, HSV and vaccinia all are manipulated to

A

target, infect and kill cancer cells

65
Q

Conditionally Replicative Adenovirus (CRAd)

A

take normal adeno
mutate it so that it prefers to grow in cancer cells
will rapidly grow and lyse cancer cells

66
Q

Modifications in adenovirus that allow tumor- specific tropism:

A
  1. Delet E1A or E1B genomic regions: thus cells only replicate in cells with dysfunctions
  2. incorporate tumor specific promoters
  3. alter virus cell attachment protein so it can cause trasnduction better in tumor cells
67
Q
  1. Delet E1A or E1B genomic regions: thus cells only replicate in cells with dysfunctions
  2. incorporate tumor specific promoters
  3. alter virus cell attachment protein so it can cause trasnduction better in tumor cell
A

modification made in the adenovirus for tumor tropism

68
Q

Benefit of modifying fiber protein to alter cell attachment on adenovirus

A

or example RGD (arginine-glycine-aspartic acid) motif mediates efficient binding to integrins that are present on many tumor cells

69
Q

Adenovirus wants to take over cell to use it’s DNA apparatus, so wants to be in a cell that is dividing a lot… what kind of cell would that be?

A

cell with p53 mutation! aka, cancer cell bc these cells are constituitvely active and adenovirus needs to use that machinary..

70
Q
  • Human telomerase reverse transcriptase (hTERT) promoter

* Highly active in

A

tumor cells like glioma, inactive in normal brain cells

71
Q

We create an adenovirus that has it’s E1A and E1B replicated and activated by ________ so that it goes to places with lots of it

A

hTERT

72
Q

Reovirus (wild-type strain) no modifications by recombinant DNA
• Inherent tumor selectivity for cells with an activated______ activity

A

Ras

73
Q

• In normal cells reovirus replication is restricted by the activation of the

A

RNA-activated protein kinase (PKR)

—tumor cells dont have this!!! thus reovirus is like, fuck yeah, i’m partying here

Immunology 3 (20 decks)

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