L17 - Gene Therapy I

Biotech Exam II

What is gene therapy?

a technique to treat a disorder by introducing, inactivating, or replacing genes in a patient’s cells instead of using drugs/surgery

Gene augmentation

involves adding a copy of a functional gene → cell with corrected function

• loss of function genes

Gene suppression

involves adding an inhibitory sequence to silence a defective gene

• gain of function genes

Genome editing

involves replacing a defective gene with a healthy copy

What are the requirements for successful gene therapy protocols?

• selecting the right gene (need to understand disease)

• identifying and accessing target cells that require treatment

• appropriate gene delivery system

• proof of principle, safety, and efficacy

• suitable manufacturing and analytical processes

Properties of somatic gene therapy:

• not inheritable

• gene expression only occurs in the target cells

• aim to cure disease only in patient, not in descendants

Properties of germline gene therapy

• inheritable

• genetic mods will be passed to the next generation

Advantages of targeting lymphocytes for somatic cell gene therapy

• relatively long lived

• readily obtainable from peripheral blood

• easy to manipulate for gene transfer

• no inactivation of gene expression during differentiation

• can be depleted post-transfer

• able to secrete large amounts of protein

• potentially useful for manipulating immune responses (CAR-T cell therapy)

Ex vivo vs in vivo

• ex vivo is done outside of body

• in vivo is done inside of body

How does ex vivo gene therapy work?

Advantages of ex vivo

• does not require tissue specific vectors

• very high transfer efficacy

• target cells can be manipulated/amplified

Disadvantages of ex vivo

• can be used for limited target cells (BCs)

• cells need to function normally post transfer

• in vitro artifacts

In vivo advantages

• can target all body tissue

• no in vitro artifacts

In vivo disadvantages

• specificity of gene transfer can be an issue

• less invasive

What are the two types of gene delivery systems?

1. viral (use of viral vectors)

2. nonviral (majority)

What are retro viruses?

• single stranded positive sense RNA viruses

• inserts a copy of its genome into the DNA of a host cell that it invades, thus changing the genome of that cell

• infection persists indefinitely

What are the components of a retrovirus

• gag gene - viral core structural proteins

• pol gene - viral enzymes for infection

• env genes - viral envelope for recognizing host cell receptors

• 2 copies of RNA genome

What is the retrovirus life cycle?

What is a provirus?

the integrated form of DNA to produce both mRNAs encoding the various viral proteins

What are the two categories of the retroviral genome?

1. Cis sequences - directly active as nucleic acids

2. trans sequences - the protein coding sequences (gag, pol, env)

What are the different components of the cis sequence?

• 5’ LTR - acts as the promoter in DNA form and contains seqs important for reverse transcription in RNA form

• PBS - first strand DNA synthesis during reverse transcription

• psi sequence - directs packaging of genomic RNA into virion

• ppt - primer binding site for second strand DNA synthesis

• 3’ LTR - in DNA acts as polyadenylation signal, in RNA important for reverse transcription

What would happen if the psi region is deleted from the viral genome?

you would have empty virus particles with no viral RNA (aka helper virus)

What would happen if you replace the trans sequence with therapeutic genes?

The resulting virus particle will contain the therapeutic gene (aka vector virus)

What could happen if you combined the helper and vector virus?

regeneration of the replication competent virus (wild type virus)

What is the main limitation of retrovirus in which all the trans sequences are replaced by the gene of choice?

they can express only one gene

What are some ways we can express multiple genes from one viral vector?

1. expression of different proteins from alternatively spliced messenger RNAs trabscribed from one promoter

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

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

Typically what is gene 1 and gene 2 in viral vectors?

gene 1 is the desired gene and gene 2 is an antibiotic resistance gene, which serves as a marker gene for molecular detection of positive target cells in the host

What are pseudotyped viruses?

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

What are advantages of using retroviral vectors?

• efficient and stable integration

• controllable host range via envelop pseudotyping

• capable of delivering up to 8 kbp of exogenous sequences

What are disadvantages of using retroviral vectors?

• can infect only dividing cells

• difficult to obtain high concentrations

• 8 kbp may not be enough

What are some safety concerns for using retroviral vectors?

• production of replication-competent virus

• insertional mutagenesis (activation of protooncogene)

What are lentiviral vectors?

• retroviruses with the ability to infect both dividing and non-dividing cells

• integrate permanently into host genome

What are some alternatives to HIV-1 based vectors?

• HIV 2 based vector - lower transmissibility

• SIV

• FIV,EIAV - non-primate based vectors

What is the number 1 vector used in gene transfer in clinical trials?

adenovirus (non-enveloped double-stranded DNA virus)

What is the adenoviral vector delivery system

1. bind to receptors and enter cell via endocytosis

2. travels to nucleus

3. DNA enters the nucleus, but does NOT integrates into genome

4. gene is transcribed and protein is produced

What is the structure of adenoviral vectors?

• early genes (E1-4) - expresses non structural, regulatory proteins

• late genes (L1-5) - viral structural protein required for viral genome packaging and assembly

• E1 and E3 are replaced with the therapeutic gene

What are limitations of first generation adenoviral vectors?

• pre-existing immunity

• leaky expression of adenoviral proteins from genes that were not deleted

  • can result in destruction of cells expressing viral proteins

What are gutted adenoviral vectors?

• have no viral protein genes present

• contains the therapeutic gene and stuffer sequences to maintain right size for packaging

• high capacity, can transduce multiple genes

• extended time of gene expression

• reduced immunogenicity

What are the main advantages of adenovirus vectors?

• large capacity of transgene

• high titers

• ability to infect a broad range of cells (dividing and non dividing)

• no evidence for chromosomal integration

• stable and does not undergo rearrangement at a high rate

• low pathogenicity

What are the main disadvantages of adenovirus vectors?

• very immunogenic

• does not integrate into host’s genome

• transient expression

What are adeno-associated viruses (AAV)?

• non-enveloped single stranded DNA virus

• requires a helper virus to produce infectious particles

What is the main problem with conventional AAV?

• slow and inefficient because the host cell must first convert the ssDNA into dsDNA for transcription to begin

What are self-complementary AAV (scAAV) vectors?

• modified vector that contains two complementary copies of the transgene arranged in an inverted orientation

• linked by mutated inverted terminal repeat (ITR) that allows for self-complementary folding

• has a reduced cargo capacity

What are the main advantages of AAV vectors?

• small, easy to manipulate

• infect both non dividing and dividing cells

• low immunogenicity

• not associated with any known human disease

What are the disadvantages of AAV vectors?

• limited packaging capacity

• requires adenovirus as helper virus

What is herpes simplex type I virus (HSV)?

• dsDNA virus

• tissue specific gene transduction

• treats CNS diseases

• can be selectively depleted by treating with ganciclovir

Comparation of different viral vectors