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53 Cards in this Set
- Front
- Back
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What are different viral vectors used in gene therapy?
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Retroviruses
Adenoviruses Adeno-ass't viruses (AAV) Herpes viruses |
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What is the aim of gene therapy?
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Correct a genetic defect by transferring of a fctnal normal copy of the gene into cells
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What is transduction?
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Delivery of a gene with a viral vector
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What does gene therapy apply to?
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Genetic disorders
Cancer: genetic disposition, mutation in oncogene or tumour suppressore gene Autoimmunity diseases: rheumatoid arthritis, delivery of counteractine gene Viral infections: delivery of DNA molec that will produce RNA or ptns vs the virus |
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What is an example of gene therapy for HIV?
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T-cell based gene therapy
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How does T cell based gene therapy work?
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Isolate CD4+ cells
Transduction of CD4+ cells with genetic vectos CD4 cell selection and expansion Infusion -->Relatively easy to obtain and genetically modify, but doesn't capture all of viral reservoirs for HIV, such as monocytes and DCs ->Only looks at CD4+ cells |
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What happens in stem cell bassed gene therapy?
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Transduction targets in this case are pluripotent stem cells or CFU-Blast
-->Target cells very early before differentiation and hematopoiesis Gene you want expressed will then be expressed in all cells .: it has a greater chance of being activated |
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What are viral vectors?
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Use virus particles to carry nucleic acids into the cells
->very efficient mechanism to do this |
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What is the rational behind viral vector use?
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Viruses are obligatory intracellular parasites
Very efficient at transferring viral DNA into host cells Target specific cells: depending on the viral attachment ptns (capsid or glycoptns) Gene replacement: non-essential genes of virus are deleted and exogenous genes are inserted |
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What are some ex of viral vectors?
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Adenovirus
Retrovirus Lentivirus AAV HSV |
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What type of virus cannot be used?
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Replication competent viruses are NOT safe and cannot be used
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What type of viruses should be used?
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Replication deficient ciruses
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Why can replication deficient viruses be used?
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Don't encode viral structural ptns
Cannot replicate beyond the 1st cycle of infection |
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What are the elements needed to generate a replication deficient viral vector?
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Transfer vector
Paackaging vector Helper virus |
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What does the transfer vector do?
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Plasmid that contains the promoter, gene of interest, Ori and packaging signal
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What does the packaging vector do?
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Large plasmid or cell lines
Provides the viral structural ptns for pkging of transder vector |
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What does the helper virus do?
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Provides additional sequences necessary for virus formation
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What is required for the biosafety of gene therapy vectors?
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Vectors must be replication defective virus particles
->Viral particles must infect cells and transport recombinant viral DNA into nucleus, but must NOT be able to produce and release new infectious particels that could transfer foreign gene(s) to other cells/tissues or from patient to another human |
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How are the viruses made to be replication defective?
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Deletion of one or more genes required for replication
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Viral vector: Adenovirus
Titer Insert size Manipulation of tropism Immunogenicity Infect non-dividing cells |
Titer: 10^11**
Insert size: 2-38 kb Manipulation of tropism: good Immunogenicity: very high (not a good thing)** Infect non-dividing cells: yes (good, can get latent infections) |
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Viral vector: Retrovirus
Titer Insert size Manipulation of tropism Immunogenicity Infect non-dividing cells |
Titer: 10^7
Insert size: 1-7kb (small) ** Manipulation of tropism: good Immunogenicity:low Infect non-dividing cells: no (Bad)** |
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Viral vector: Lentivirus
Titer Insert size Manipulation of tropism Immunogenicity Infect non-dividing cells |
Titer: 10^7
Insert size: 7-18kb Manipulation of tropism: good Immunogenicity: low Infect non-dividing cells: yes |
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Viral vector: AAV
Titer Insert size Manipulation of tropism Immunogenicity Infect non-dividing cells |
Titer: 10^7
Insert size: 4.9kb (small) ** Manipulation of tropism: not so good Immunogenicity: low Infect non-dividing cells: yes |
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Viral vector: Herpesvirus
Titer Insert size Manipulation of tropism Immunogenicity Infect non-dividing cells |
Titer: 10^7
Insert size: 50kb (huge)** Manipulation of tropism: not so good (mostly in neuronal cells) Immunogenicity: low Infect non-dividing cells: yes |
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What is an ex of a retroviral vector?
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Moloney murine leukemia virus (MuLV)
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What would the MuLV transfer plasmid vector contain?
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Gene of interest
LTR: promoter, poly A, integration, replication and revT Primer binding site (PBS): origin of replication RNA packaging signal Polypurin tract (imp for revT) |
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What would the packging vector of MuLV have?
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Cell line stably transfected with plasmid contructs containing Gag, Gag-Pol and Env
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How do you make a retroviral vector?
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Insert the transgene INTO the Gag sequence (.: can't make gag or the other genes)
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Describe the retroviral vector particle
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Have a packaging cell line that expresses viral structural ptns, but no packageable viral RNA (so you can get a viral structure without a genome)
Aff a transfer vector plasmid containing the foreign gene |
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How are retroviral vectors produced?
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1)Retroviral vector plasmd
2) Packaging cell line: if you only use this, get an empty vector, just have capsid ptns + pol enz 3)Make infectious replication defective RV (only transgenee is expressed, Gag-pol-env cannot be T .: only get 1 cycle of replication so can't infect other cells) 4) Transduce target cell |
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What are the issues with retroviral gene therapy?
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1) Regeneration of replication competent RVs
2) Insertional mutagenesis: carcinogenic 3)Limited encelope tropism 4)Heterologous gene expression wanes with time 5) RVs, except for lentiviruses, do not infect non-dividing cells |
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How can there be a regeneration of a replication competent RV?
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Homologous and non-homologous recombination events have led to the rescue of replication compentent RV
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How can this problem be solved?
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Modify LTR and insert a non-retroviral promoter (CMV) .: can't recombine
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How can the insertion of an RV into the genome be carcinogenic?
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RV can integrate into the genome
If close to a protooncogene, can activate it |
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How can limited envelope tropism be overcome?
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"Pseudotyped" vector particles with particles with pantropic Env, like VSV-G ptn (can change Env ptns)
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What type of vector can be used to deliver genes into a terminally differentiated cells?
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Use lentiviral vectors
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What are the better vectors to use?
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Small antiviral vectors
->Can integrate, might not activate proto-ncs |
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Describe lentiviral vectors
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Keep 5' LTR, Express B'globin, have the central polypurine Tract and the woodchuck post-T element
Should be transfected with viral trans fcts Gag, Pol, Env, Tat and Rev using ,ultiple separate plasmids |
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Describe the adenoviral vector
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Replication defective forms of human adenovirus type 5 most common
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Where does natural Ade5 infection occur?
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In young children, with no/mild respiratory illness
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Describe the Ade5 vector
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Essential E1 genes are deleted
cDNA + promoter/enhancer seq inserted in place of E1 or non-essential E3 genes |
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Where is recombinant DNA genome transfected into?
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E1 producing 293 cells to recover recombinant viral particles
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What do the new generations of adenoviral vectors have?
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They are safer and can have bigger vectors
Have loxP: can cleave itself It is also less immunogenic |
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What are the adv of adenoviral vectors?
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High titers
Both dividing and non-dividing cells Wide tissue tropism Can modify viral pentons to alter tissue tropism |
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What are the disadv of adenoviral vectors?
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Transient expression (not good for genetic diseases)
Highly immunogenic (because not integrated) High titers can be toxic Gene T with E1-neg virus is leaky: many genes expressed at low lvl CD8 CTL responses to these processed adenoviral peptides leads to elimination of infected cells expressing foreign genes |
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Are the new adenoviral genomes that lack most of the genes better?
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Yes, but need a special packaging cell lines
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Describe AAV vectors
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ssDNA
Parvovirus Small: 4.5 kb |
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How does this virus need to be transfected?
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Needs to be co-transfected with plasmids that have Rep and Capsid ptns into the 293 cell line where packaging occurs with assitance of adenoviral helper ptns into replication defective AAV particles
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What are the disdv of AAV vector?
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Insert length restricted to 4.9kb
May integrate into host genomic DNA :pw titer of virus, low lvl of gene expression |
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How are AAV vectors generated?
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1) Remove Rep and Cap. Insert Transgene and maybe polyA
2)AAV packaging plasmid with Rep and Cap required 3) Also need AAV helped plasmid (has E1A/B, VA, E2A, E4) |
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What are the adv of AAV vectors?
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Integration and persistent expression
No insertional mutagenesis Infecting dividing and non dividing cells Safe |
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Describe Herpesvirus vectors
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HSV-1 causes mild disease
dsDNA 50kb of foreign DNA can be inserted (150kb for the new generation of vectors) Neurotropic virus (brings things to the neurons) Highly deleted defective virus (half of the 80 HSV genes can be deleted) + packaging of cell lines with mutant strains |
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What has the HSV been used as a vector for?
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To express shRNAs vs SV40 LT Ag
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