Lecture 17: Gene Therapy Viral

somatic gene therapy

• non inheritable

• gene expression only occurs in target cells

• aim to cure a disease only in patient

germline gene therapy

• inheritable

• gene modification of germ cells

somatic

only __ gene therapy has been tested in humans

ex vivo

• does not require tissue specific vectors

• very high transfer efficiency

• target cells can be manipulated

ex vivo

• can be used for limited target cells

• cells need to retain ability to “home” and function normally post-transfer

• in vitro artifacts

in vivo

• can target all body tissues

• no in vitro artifacts

in vivo

• specificity of gene transfer

• less invasive

viral gene delivery systems

• retroviral AND lentiviral

• adenoviral

• adeno-associated viral

• herpes simplex viral

nonviral gene delivery systems

• liposomes

• nanoparticles

• biolistic gene gun

• naked DNA

retroviruses

• single-stranded positive-sense RNA viruses

• inserts copy genome into DNA of host cell it invades

gag gene products (viral core structural proteins)

• matrix (MA)

• nucleocapsid (NC)

• capsid (CA)

pol gene products (viral enzymes)

• reverse transcriptase (RT)

• integrase (IN)

• protease (PR)

env gene products (viral envelope)

• surface subunit (SU)

• transmembrane subunit (TM)

cis sequences

1. 5’ long terminal repeat (LTR)

2. primer binding site (PBS)

3. psi sequence

4. polypurine tract (ppt)

5. 3’ LTR

trans sequences

1. protein-coding genes (gag/pol/env)

5’ long terminal repeat (LTR)

DNA: found in provirus as transcriptional promoter

5’ long terminal repeat (LTR)

RNA: contains sequences important for reverse transcription of genome

primer binding site (PBS)

first strand DNA synthesis during reverse transcription

polypurine tract (ppt)

primer binding site for second strand DNA synthesis during reverse transcription

3’ long terminal repeat (LTR)

DNA: acts as polyadenylation signal

3’ long terminal repeat (LTR)

RNA: sequences important for reverse transcription of genome

no viral RNA packaged

result of psi sequence deletion

first generation helper construct

• contains gag/pol/env → make viral proteins

• psi sequence depleted

first generation vector construct

• contains therapeutic gene

• keeps LTRs and psi packaging signal

• RNA packaged into viral particles

replication competent virus

first generation retroviral vector systems are risky since they can generate __

second generation helper construct

• separates gag/pol and env

• psi sequence depleted

replication incompetent vector virus

• result of second generation packaging system

• infects once but cannot replicate

simplest retroviral vector

all trans sequences (gag/pol/env) replaced by only gene of choice and can only express one gene

splicing vector

expression of different proteins from alternatively spliced messenger RNAs transcribed from one promoter

internal ribosome entry site vector

use of IRES elements to allow translation of multiple coding regions from a single mRNA

internal promoter vector

use of promoter in the LTR and internal promoters to drive transcription of different cDNAs

desired gene

gene 1 from vectors

antibiotic resistance gene (marker gene)

gene 2 in vectors

vector virus

infects natural host cell populations

pseudotype vector virus

envelope proteins consist of parts of viral protein necessary for incorporation into virion and sequences meant to interact with specific host cell proteins

dividing

retrovirus can only infect __

insertional mutagenesis

possibility if retroviral DNA integrates near wrong gene and activates protogene

lentiviral vectors

• more complex type of retroviruses

• ability to infect both dividing and non-dividing cells

• integrate permanently into host genome

• explored for HIV-1 and HIV-2

Adenoviral Vector Delivery System

• non-enveloped double-stranded DNA virus

• for mild respiratory infections & other illnesses

endocytosis

process by which adenoviral vectors enter

first generation adenoviral factor

• early genes (E1-E4)

• late genes (L1-L5)

early genes (E1-E4)

express non-structural, regulatory proteins

late genes (L1-L5)

viral structural protein required for viral genome packaging and assembly

pre-existing immunity, leaky expression

limitations of adenoviral vector

“gutted” adenoviral vector

• no viral protein genes present

• high capacity adenoviral vector

• extended time of gene expression

• reduced immunogenicity

Adeno-Associated Viral (AAV) Genome parts

ITRs, replication, capsid

Recombinant Vector Genome parts

ITRs, promoter, therapeutic gene, poly A

Self-Complementary AAV parts

ITRs (both ends), promoter, transgene, polyA signal

polyA signal

ensures proper transcription termination

promoter

drives expression of gene

Herpes Simple Type 1 Virus (HSV)

• double-stranded DNA virus

• treat CNS (Parkinson’s)

HSV Thymidine Kinase (HSV-TK)

can activate Ganciclovir to kill cell