gene therapy I

what is gene therapy?

• a technique that allows doctors to treat a disorder by modifying a gene in a patient's cells instead of using drugs/surgery

• introducing a new gene into the body to help fight a disease

• inactivating or "knocking out" a mutated gene that causes disease with a healthy copy of the gene

• involves the transfer of genetic material into host cells to cure or favorably modify the clinical course of a disease

what are the applications of gene therapy?

• replacement of defective genes: e.g. CFTR gene in CF
• supplying a gene product in large quantities in an unregulated manner: e.g. factor IX in hemophilia
• introducing genes into a cell that do not normally express them: e.g. "immunopotentiation/suicide genes" in cancer cells
• introducing protective genes into target cells: e.g., viral-specific ribozymes to treat HIV-1 infections
• modulation of immune response: e.g. tolerance induction in autoimmune diseases
• DNA vaccinations: e.g. West Nile virus DNA vaccine for vet applications

what are the requirements for successful gene therapy protocols?

• selecting the right gene: understand the disease
• identifying and accessing target cells that require the treatment
• appropriate gene delivery system
• proof of principle, safety, and afficacy
• suitable manufacturing and analytical processes

what are the target cells of somatic gene therapy?

• not inheritable
• gene expression only occurs in the target cells
• aim to cure a disease only in the patient, not in the patient's descendants

what are the target cells of germline gene therapy?

• inheritable
• genetic modification of germ cells that will pass the selected changes to the next generation
• used in the form of transgenic/"knock out" animals" in which an exogenous gene is either introduced/deleted

what are the 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, a useful safety back-up
• can be used to supply blood-borne gene products: able to secrete large amounts of protein: hemophilia B (factor IX)
• potentially useful for manipulating immune responses: Car-T cell immunotherapy

what is ex vivo vs in vivo gene therapy?

ex vivo:

• gene transfer to host cell
• engineered cells containing therapeutic gene: lymphocytes, bone marrow cells, liver cells, skin cells
  in vivo:
• injectable vector

what is the process of ex vivo gene therapy?

1. remove patient X cells

• gene transfer

1. some cells now X+

• select X+ cells

1. amplify X+ cells
2. return genetically modified cells to patient

what is in vivo gene therapy?

gene transfer into target cells occurs in patient body

what are the advantages of ex vivo gene therapy?

• does not require tissue-specific vectors
• very high transfer efficiency
• target cells can be manipulated/amplified

what are the disadvantages of ex vivo gene therapy?

• can be used for limited target cells such as blood cells
• cells need to retain the ability to "home" and function normally post-transfer
• in vitro artifacts

what are the disadvantages of in vivo gene therapy?

• specificity of gene transfer can be an issue
• less invasive

what are the advantages of in vivo gene therapy?

• can target all body tissues
• no in vitro artifacts

what is the process of gene delivery systems of harmless viruses?

1. remove virus' own genes and replace with the gene of interest
2. package the gene of interest into viral particles to "smuggle" genes into target cells simply by infecting the target cell
3. when used to transfer genes into target cells, the viruses are called viral vectors
4. some viral vectors not only carry genes into the cell, but integrate (insert) the gene into host cell chromosomes

what are examples of viral and nonviral gene delivery systems?

viral:

• retroviral and lentiviral
• adenoviral
• adeno-associated viral
• herpes simplex viral
  nonviral:
• liposomes
• nanoparticles
• biolistic gene gun
• naked DNA

what are the major vectors used in gene therapy clinical trials?

adenovirus, retrovirus, plasmid DNA, lentivirus

what are retroviruses used for gene delivery?

• single-stranded (+)-sense RNA viruses
• a type of RNA virus that inserts a copy of its genome into the DNA of a host cell that it invades, thus changing the genome of that cell
• the infection will persist indefinitely

what are the components of a retrovirus particle?

• 2 copies of RNA genome
• viral core structural proteins: gag gene products
• viral enzymes: pol gene products for viral infection
• viral envelope: env gene products that dictate tropism and are for recognizing specific receptors on the host cell and initiating the infection process

how does the right RNA get packaged into the virion particle?

RNA → dsDNA → RNA → polypeptide

what is a provirus?

the integrated form of DNA to produce both mRNAs encoding the various viral proteins, and the genomic RNA that is packaged into progeny virions

what are cis sequences of the retroviral genome?

directly active as nucleic acids

what are the components of cis sequences of the retroviral genome?

• 5' long terminal repeat (LTR): in the DNA form found in the provirus acts as a transcriptional promoter, and in the RNA (genomic) form contains sequences important for reverse transcription of the genome
• primer binding site (PBS): for first-strand DNA synthesis during reverse transcription
• psi sequence: directs packaging of the genomic RNA into the virion
• polypurine tract (ppt): primer binding site for second-strand DNA synthesis during reverse transcription
• 3' LTR: in the DNA form, acts as a polyadenylation signal, and in the RNA genomic form, contains sequences important for the reverse transcription process

what is the trans sequence of retroviral genomes?

the protein-coding sequences

what happens when the packaging signal psi is deleted from the viral genome?

• empty virus particles
• replication of defective vectors

what are replication-defective retroviral vector systems?

• separating the cis and trans genetic functions
• vector construct: contains cis sequences
• helper/packaging plasmids: encodes the viral proteins
• made by separating the cis and trans functions of the virus into a vector construct, which contains the cis sequences, and helper or packaging plasmids that encode the viral proteins

what is the simplest first generation retroviral vector system?

• directs packaging of the genomic RNA into the virus
• recombination between helper and vector virus could regenerate a replication-competent virus

what is the process of vector virus production and infection?

1. packaging/helper cell
2. replication-incompetent vector virus
3. gene delivery into target cells by infection

what is the simplest retroviral vector

• a retrovirus in which all the trans sequences (gag/pol and env) were replaced only by the gene of choice
• the main limitation of such vectors is that they can express only one gene

what are strategies to express multiple genes from one viral vector?

• expression of different proteins from alternatively spliced messenger RNAs transcribed from one promoter: splicing vector; transcription of the second gene is much less efficient
• use of IRES elements to allow translation of multiple coding regions from a single mRNA: internal ribosome entry site vector
• use of the promoter in the LTR and internal promoters to drive transcription of different cDNAs: internal promoter vector

what are the multiple genes of a viral vector?

• gene 1: desired gene
• gene 2: an antibiotic resistance gene that serves as a marker gene for molecular detectionnof (+) target cells in the host

what are the strategies to expand host range?

• viral vectors have natural host cell populations that they infect most efficiently, which limits natural host cell ranges
• one of the purposes of gene therapy might either want to limit or expand the range of cells susceptible to transduction by a gene therapy vector
• pseudotyped viruses: the envelope proteins consist of the parts of the viral protein necessary for incorporation into the virion and the sequences meant to interact with specific host cell proteins

what are the advantages and disadvantages of using retroviral vectors?

advantages:

• efficient and stable integration
• controllable host range via envelope pseudotyping
• capable of delivering up to 8 kbp of exogenous sequences
  disadvantages:
• can infect only dividing cells
• difficult to obtain high titers (i.e. high concentrations)
• 8 kbp may not be enough

what are safety concerns for using retroviral vectors?

• production of replication-competent virus: separate gag-pol and env genes such that assembly of the entire viral genome (cis and trans sequences) requires 2 non-homologous recombination events, which has a very low probability of occurring
• insertional mutagenesis: integration can activate a protooncogene and has been reported only in IL2RG gene therapy trials thus far

what are lentiviral vectors?

• more complex type of retroviruses
• ability to infect both dividing and non-dividing cells
• integrate permanently into the host genome
• human lentiviruses that have been explored for gene delivery systems are HIV-1 and HIV-2

what is the skepticism with HIV-1-based vectors?

• HIV-1 is. ahuman pathogen in nature
• there is risk of generating wild-type HIV-1 during vector virus production

what are alternatives to lentiviral vectors?

• HIV-2-based vectors: ~50% divergent from HIV-1; accounts for only 1% of HIV infections as it has lower transmissibility
• SIV (simian immunodeficiency virus): species of retrovirus that causes persistent infections in at least 45 species of African non-human primates
• non-primate lentivirus-based vectors: FIV (feline immunodeficiency virus), EIAV (equine infectious anemia virus)

what are the most used vectors in gene therapy clinical trials?

adenovirus (14.7%)

what is the adenoviral vector delivery system?

• non-enveloped double-stranded DNA virus
• mild respiratory infections and other illnesses
• therapeutic gene extrachromosomal DNA does not integrate

what is the process of the adenoviral vector delivery system?

1. attachment and entry

• binds to receptors on the surface
• enters via endocytosis

1. escape and transport

• escapes endosome
• travels to the nucleus

1. gene delivery

• DNA enters nucleus and does not integrate into genome

1. expression

• host machinery transcribes the delivered gene → protein produced

what are adenoviral vectors?

• first generation: ΔE1 and ΔE3
• high titers and ability to infect a broad range of non-dividing and dividing cells**
• the early genes (E1-E4): express non-structural, regulatory proteins
• the late gene products (L1-L5): viral structural protein required for viral genome packaging and assembly
• can be replication-defective
• certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene

what is the limitation of the first-generation adenoviral vectors?

• pre-existing immunity to the vector virus, and their immune system attacked the viral vector
• leaky expression of adenoviral proteins from genes that were not deleted in the first-generation vectors
• adenoviruses are strongly immunogenic

what is the process of immune response to adenoviral vectors?

1. packaged into vector virus with adenoviral packaging cells
2. infect target cells
3. leaky expression adenoviral proteins
4. sustained immune reaction
5. destroy cells expressing viral proteins

what are improved adenoviral vectors?

• "gutted": no viral protein genes present
• contain stuffer sequences: to maintain right size for packaging
• high capacity: multiple genes can be transduced in one shot
• extended time of gene expression
• reduced immunogenicity
• limited number of repeat treatment with adenoviral vector

what are the advantages of adenovirus vectors?

• large capacity of transgene (36kb)
• high titers
• ability to infect a broad range of cells
• infect both dividing and non-dividing
• number of evidence for chromosomal integration
• viral genome stable and does not undergo rearrangement at a high rate
• low pathogenicity of virus in humans

what are the disadvantages of adenovirus vectors?

• very immunogenic
• do not integrate into the host's genome
• transient expression: could be good for modulated immune response, developing vaccines, and cancer treatment

what is adeno-associated virus (AAV)?

• non-pathogenic, defective virus that requires a helper virus to supply the machinery for producing infectious particles (adenovirus helper virus, rep/cap)
• AAV vectors: non-enveloped single-stranded DNA virus
• main point of consideration in the rational design of an rAAV vector: packaging size of the expression cassette that will be placed between the 2 ITRs

what is the problem with conventional AAV (ssAAV)?

• package single-stranded DNA
  after infection:
• virus enters the cell and delivers ssDNA into the nucleus
• the host cell must convert ssDNA to double-stranded DNA (dsDNA)
• only then can transcription begin
• the second-strand synthesis is often slow and inefficient, especially in: non-dividing cells or certain tissues (e.g. muscle, neurons)

what are self-complementary AAV (scAAV) vectors?

• the genome contains 2 complementary copies of the transgene arranged in an inverted orientation
• linked by a mutated inverted terminal repeat (ITR) that acts like a hinge

what are the key components of self-complementary AAV (scAAV) vectors?

• promoter: drives expression of your gene (e.g. CMV, CAG)
• transgene: gene of interest (must be small due to size limit)
• polyA signal: ensures proper transcription termination
  ITRs (inverted terminal repeats):
• located at both ends
• required for replication and packaging
• one ITR is modified to allow self-complementary folding

what is the trade-off for self-complementary AAV (scAAV) vectors?

• reduced cargo capacity
• scAAV can carry ~2.2-2.5 kb vs ~4.7 kb for single-stranded AAV

what is designing new AAV vectors?

• having tem0porally modulatable expression
• use a muscle-specific promoter to drive gene expression
• can target the viral vector to specific cells using a designer Cap protein
• can use bidirectional promoters to drive multiple gene expression

what are the advantages and disadvantages of AAV vectors?

advantages:

• small, easy to manipulate virus
• infect a wide range of both non-dividing and dividing cells
• low immunogenicity
• not associated with any known human disease
  disadvantages:
• limited packaging capacity (~5kb)
• requires adenovirus as helper virus, although producer cell lines have recently been developed

what is herpes simplex type I virus (HSV)?

• double-stranded DNA virus
• neutropic virus: tissue-specific gene transduction
• treat CNS diseases such as Parkinson's
• can be selectively depleted by treating with ganciclovir: virus naturally encodes HSV thymidine kinase (HSV-TK) gene