6 - Gene Therapy

Hemophilia B (Christmas Disease)

- X-linked genetic disorder - affects 1/100,000 males births - Abnormal bleeding due to defective coagulation factor IX - Mutation in factor IX known to be cause of "royal disease 40% of cases are severe in factor IX activity Effective care can be achieved w/ levels of 75-100 ng/ml factor IX in blood - Symptoms can be significantly reduced by only 1.5-2% of normal levels - Factor IX cDNA gene - 1.5kbp

Safety Concerns for Retroviral Vectors

1) production of replication competent virus - Separate gag-pol and env genes such that assembly of the entire viral genome (cis and trans sequences) requires two non-homologous recombination events - extremely low probability 2) insertional mutagenesis - Integration can activate a protooncogene - Has been reported only in IL2RG gene therapy trials thus far

how to express multiple genes from viral vector

1. Expression of different proteins from alternatively spliced messenger RNAs transcribed from one promoter - Transcription of second gene is much less efficient - Sequence b/w the two genes - Two mRNA species produced: one translating only gene 1, one translating only gene 2 2. Use of Internal Ribosome Entry Site Vector (IRES) to allow translation of multiple coding regions from a single mRNA - One stretch of DNA includes IRES - allows gene 2 to also be made 3. Use of promoter in the LTR and internal promoters to drive transcription of different cDNAs - Internal Promoter Vector - Use a separate promoter for gene 2 - 2 genes driven by 2 different promoters - Allows most suitable promoter to be chosen for the second gene Expression of multiple genes from single virus vector may benefit gene therapies: increase number of gene products per virus vector delivery of distinct genes

Improved targeting of liposomes

1. PEGylated liposomes - PEG for improved pharmacokinetics and pharmacodynamics 2. Transferrin-Conjugated Liposome - transferrin can bind transferrin receptors found on human cells 3. Antibody-modified liposomes - antibody coating for specificity 4. Sendai (HVJ) viral proteins allows DNA to escape endosome intact by disrupting endosomal membrane 5. DNA binding proteins (HMG-1 high mobility group 1 protein) to target DNA into nucleus 6. Nuclear Localizing Sequence (NLS) within DNA Wide range of DNA encoding both small or large proteins can be accommodated, liposomes offer versatile option for gene delivery After internalization of the liposome the most challenging step is release of the genetic construct from the endosome to the cytoplasm Liposome content should be able to escape from the endosome and be free

Non-viral systems

25% of gene delivery systems examples - liposomes - nanoparticles - biolistic gene gun - naked DNA

X-linked severe combined immunodeficiency (SCID)

40-50% of SCID cases Caused by deficiency in gamma subunit of cytokine IL-2 receptor (IL2RG gene) Gamma subunit of IL-2 receptor also called common gamma chain - subunit to receptors for five other cytokines - IL-4, 7, 9, 15, 21 4/10 patients in gene therapy developed leukemia Possible reasons for leukemia development - Something about vector virus made it integrade at oncogenic sites (LMO2) - Transferred gene IL2RG itself is oncogenic - Genetic background of patient makes them predisposed to cancer Ongoing retroviral vector trial at Boston Children's Hospital

Viral Systems

75% of gene delivery systems harmless viruses 1. remove virus' own genes and replace w/ gene of interest 2. package gene of interest to smuggle genes into target cells simply by infecting the target cell 3. When used to transfer genes into target cells - 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 examples - retroviral and lentiviral - adenoviral - adeno-associated viral - herpes simplex viral adenovirus + retrovirus are most common vectors used

Advantages and Disadvantages of Retroviral Vectors

ADV - efficient and stable integration - controllable host range via envelope pseudotyping - capable of delivering up to 8 kbp of exogenous sequences DIS - can infect only dividing cells - can only enter nuclear membrane after it has dissolved - lentiviruses - viral DNA can enter nucleus using carrier proteins - difficult to obtain high titers - high concentrations - 8 kbp may not be enough

ADA-SCID

Adenosine deaminase deficiency ADA deficiency is cause of approximately 20% of cases of severe combined immunodeficiency (SCID) Patients have multiple severe opportunistic infections beginning in infancy Inherited as an autosomal recessive condition Rare - less than 1/100,000 births Usually fatal in first year of life if untreated Selective toxicity to lymphocytes from accumulation of metabolites of deoxyadenosine, especially dATP

Advantages/Disadvantages of In Vivo Gene Therapy

Advantages - no in vitro artifacts Disadvantages - can target all body tissues - specificity can be an issue - less invasive

Advantages/Disadvantages of Ex Vivo Gene Therapy

Advantages - vectors used do not have to be tissue specific - efficient DNA transfer - target cells can be easily manipulated/amplified Disadvantages - only can be used for limited target cells such as blood cels (lymphocytes and bone marrow) and liver and skin cells - cells need to retain ability to home and function normally post transfer - in vitro artifacts - may introduce something abnormal from the in vivo study

Advantages and Disadvantages of Adenoviral Vectors

Advantages Large capacity of transgene High titers Ability to infect broad range of cells Infect both dividing and non-dividing No evidence for chromosomal integration Viral genome stable and does not undergo rearrangement at high rate Low pathogenicity of virus in humans Disadvantages Very immunogenic - Many viral genes that produce immunogenic proteins are cis-acting - Still remains in vector - trying to minimize number of immunogenic genes included - not entirely solved Does not integrate into host genome Transient expression - Can be an advantage: - Modulate immune response - Develop vaccines - Cancer treatment

Advantages/Disadvantages of AAV Vectors

Advantages Small, easy to manipulate virus Infect a wide range of non-dividing and dividing cells Low immunogenicity Not associated w/ any known human disease Disadvantages Limited packaging capacity (5kb) Requires adenovirus as helper virus (some producer cell lines have been developed though)

Retrovirus Life cycle

Attachment then entrance Once in the host cytoplasm - virus uses reverse transcriptase enzyme to produce DNA from its RNA genome New DNA then incorporated into host cell genome by integrase enzyme Retroviral DNA once integrated - provirus Host cell then treats viral DNA as part of its own genome - transcribes and translates the viral genes along w/ the cells own genes producing the proteins to assemble new copies of the virus Difficult to detect virus until it has infected host but once infected the infection will persist indefinitely Important point in life cycle of virus that make them uniquely able to transfect genes - integrate into the host genome - Can infect a wide variety of cells b/c use commonly expressed membrane proteins at the receptors

Parkinson's Disease

Cell death in substantia nigra Responsible for making neurotransmitter dopamine Currently treated w/ exogenous dopamine systemically which inefficiently crosses BBB and has side effects Gene therapy - gene transduction of tyrosine hydroxylase - enzyme that synthesizes dopamine

Cancer Vaccine

Collect tumor cells Engineer w/ genes that cause them to be more conspicuous to the immune system Re-infusing altered cells into patient w/ immune-stimulating compound Patient's immune system launches vigorous attack not only on newly infused cells but on similar cancer cells throughout body

Why gene therapy needed for cardiac disease

Coronary arteries re-narrow after stenting Endovascular surgery - less invasive procedure used to treat problems affecting the blood vessels - restenosis is the common adverse event

Advantages of Targeting lymphocytes for somatic cell gene therapy

Easy to obtain Blood-borne Long-lived Much is known about their biology Able to secrete large amounts of protein Potentially useful for manipulating immune responses

Nanoparticles for gene delivery - Polymer-based nanomaterials for gene delivery

Electrostatic interactions w/ DNA and polymer allow nanocomplexes to be formed w/ DNA protecting DNA from degradation Poly-glycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) are approved as polymers Can potentially be used for gene delivery Advantages Increased transfection efficiency Can navigate through multiple biological barriers

Gene therapy in cancer

Engineer T-cells to recognize tumor antigens (CEA, NY-ESO-1, CD19) Immunopotentiating genes (IL2, B7, GM-CSF) introduced into tumor cells to increase immune reaction to tumor Restoring tumor suppressor genes (p53, BRCA1, Rb) that are mutated in cancer cells Antisense therapy ot turn of oncogenes/replication genes Suicide genes - implant herpes simplex thymidine kinase into tumor cells

CAR T-cell therapy

Enhance natural cancer fighting ability of patient's own T-cells Sequence: - Patients T cells collected and mixed w/ viruses carrying several specific genes - Viruses deliver these genes to T cells nuclei where they're incorporated into the T cell DNA - Genes cause the T cells to express special protein called chimeric antigen receptor - CAR on surface - CAR directs T-cell to tumor using specific location and CAR T cell is equipped to rapidly destroy cancer cell - When CAR T cells are infused into patient - seek out tumor cells and proliferate to make more cancer-killing cells

Gene therapy

Experimental technique that uses genes to treat or prevent disease May allow doctors to treat a disorder by inserting gene into patient's cells instead of using drugs or surgery Approaches - Replacing mutated gene that causes disease w/ healthy copy - Inactivating or knocking out a mutated gene that is functioning improperly - Introducing new gene into the body to help fight a disease Transfer of genetic material into host cells to cure or favorably modify clinical course of a disease

Long-term expression of Factor IX after gene therapy using AAV

Expression up to 11% of the normal protein AAV - single stranded DNA - Designed AAV vector - Contained single strand of DNA encoding two complementary copies of the factor IX gene - copies are self-complementary - Vector called sc-AAV Complementary strands allows to overcome rate limiting step in AAV life cycle - 2nd strand DNA strand - When infect target cell - don't have to wait for pieces of the second strand - Instead the two strands snap together to form ds DNA ready for gene expression Complementary sequences resulted in significantly higher gene transfer efficiency Reduced capacity of AAV by half - Maximally only 2.3-2.4 kbp in size

Adenoviruses are Strongly Immunogenic

First gen only had e1/e3 deleted - had limitations in in vivo clinical trial gene therapy - Caused strong immunogenicity Adenoviruses are common human viruses - Some patients had pre-existing immunity to those viruses - Immune system attack viral vector - Causes poor gene transfer

Gene Therapy for CF

First trials in 1993 used adenoviral vectors - unsuccessful due to significant immune reactions and inflammation in the lungs Clinical trial currently ongoing in UK - CFTR gene introduced via aerosol to lungs using cationic liposomes - Some assigned nebulized gene therapy or saline (placebo) at months intervals over a year - Lung function evaluated using common clinical measure FEV1 Patients who received therapy had significant benefit in lung function - FEV1 was larger after 1 year of treatment First trial to show that repeated doses of a gene therapy can have meaningful effect on the disease and change lung function of patients

Improved Adenoviral Vectors (Gutted)

Gutted adenoviral vector - no viral protein present - Only can change gene of interest If gene of interest is less than 36 kbp - may be stuffer sequences included - Stuffer sequences maintain right size for packaging Like retroviral vectors, gutted adenoviral vectors require packaging cells to supply viral proteins in trans Advantage of gutted - High capacity adenoviral vector - multiple genes can be transduced in one shot - Extended time of gene expression - Reduced immunogenicity - Limited repeat treatment

Cis sequences

HAVE to be present in genome backbone Two LTRs - long term repeats - act as transcriptional promoters in DNA form, in RNA form contains sequences important for reverse transcription of the genome - drive expression, reverse transcription, etc. - PBS - primer binding site for first DNA strand synth during reverse transcription - Psi sequence - packaging of genomic RNA into virion - polypurine tract ppt - primer binding site for second strand RNA synth during reverse transcription - 3' LTR - DNA form acts as polyadenylation signal and in RNA form contains sequences important for the reverse transcription process

Skepticism w/ HIV-1 based vectors and alternatives

HIV-1 is severely pathogenic in nature - Risk of generating wild-type HIV-1 during vector virus production Alternatives - HIV-2 based vectors (HIV-2 is 50% divergent from HIV-1), accounts for only 1% of HIV infections and has lower transmissibility - SIV (simian immunodeficiency virus) and non-primate lentivirus based vectors (FIV, EIAV)

Suicide Gene therapy advantages/disadvantages

Induces tumor lysis by tumor-specific delivery of prodrug-activating genes Has shown limited clinical efficacy for treatment of malignant glioma Advantages Requires short term gene expression Safety in clinical trials Bystander tumor killing via gap-junction delivery of drug Disadvantages Limited spatial distribution of gene transfer vectors Poor gene transfer efficiency into tumor cells in vivo Inability to target dispersed tumor cells

Current Treatment for Hemophilia B

Infusion of factor IX protein IV Concentrated from human blood Improved manufacturing and production of Factor IX has reduced complications of contaminations associated w/ using a blood product Factor IX half life - 18-24 hours In america - infusions given usually only in management of crisis situations Can cost up to 20 mill over the lifetime of the patient Gene therapy which obviates IV infusions would advance care clinically and economically

Crispr-Ca9 engineering of T cells in cancer patients

Isolated T cell from cancer patient Loaded crispr cas9 complex w/ guide RNA and electroporated into the T cell TRAC, TRBC, PDCD1 all edited Cells transduced w/ lentiviral vector to express TCR specific for cancer Then IV infused

Example of failed clinical trial

Jesse Gelsinger - OTC deficiency (ornithine transcarbamylase deficiency) Adenovirus administered to hepatic artery in liver Vector invaded organs other than intended target Only 1% of transferred genes reached target cells Harsh immune response was triggered - May be undetected genetic condition or latent Parvovirus infection

Hereditary blindness

Leber's Congenital Amaurosis (LCA) Caused by mutation in RPE65 gene Eye disorder primarily affecting retina 4 independent clinical trials: - AAV2-containing RPE65 - Reported improvement in vision - No vector-related adverse events

Lentiviral Vectors

More complex type of retroviruses Ability to infect both dividing and non-dividing cells - Penetrate even in terminally differentiated cells - Neurons, muscle, liver cells Integrate permanently into host genome

Lysosomal Storage Disease

More than 50 diseases are considered lysosomal storage disorders Most common - Gaucher disease, Fabry disease, Hunter syndrome, Hurler syndrome, Tay-Sachs disease, Pompe disease Rare, inherited conditions occur when body doesn't produce the enzymes needed to help break down certain substances such as fats sugars or proteins

Hemophilia A

Most common type of hemophilia 5x more common than hemophilia B X-linked disorder Factor VIII gene near Factor IX on X chromosome Gene (cDNA) is 9kbp Good target for gene therapy as even a low level of secreted protein expression can prevent symptoms Factor VIII is larger than IX - scAAV can't be used - max size is 2.2-2.4kbp Must try other gene delivery systems in hemophilia A

Naked DNA

Muscle, liver, skin have been shown to take up naked DNA and expressed proteins 99% of injected DNA is degraded by extracellular nucleases Large amounts of DNA required but is not immunogenic Very low transfer efficiency leads to low and transient protein expression Useful when effect of expressed protein is naturally amplified - ex. to activate immune system via cytokines or antigens Can increase transduction efficiency by electric impulse or gene gun

Adeno-Associated Viral (AAV) Vectors

Non enveloped single-stranded DNA virus Non-pathogenic, defective virus that requires a helper virus to supply machinery for producing infectious particles Main point of consideration is that rational design of rAAV vector is packaging size of expression cassette that will be placed b/w the two ITRs

Adenoviral Vector Delivery System

Non-enveloped double-stranded DNA viruses - Cause mild respiratory infections + other illnesses Part of the genome can be removed and inserted in trans - Then replaced in viral backbone w/ genes of interest Viral genome of adenoviruses does not integrate into host genome Adenoviral genome exists as extrachromosomal DNA Eliminates risk of insertion of mutagenesis Extrachromosomal DNA is not replicated when host cell divides - Daughter cells don't contain transgenes Gene delivery into growing cells w/ adenoviruses is transient and may require readministration

Considering ADA-SCID for gene therapy trials

Normal human ADA gene had been cloned Single gene defect leads to loss of enzymatic function HLA-matched allogeneic bone marrow transplantation completely corrects the disease Treatment for those patients who lack HLA-matched bone marrow donors is with recombinant ADA protein Corrected cells have selective growth advantage in vivo Small amount of ADA activity sufficient to correct disease

Clinical applications of gene therapy

Parkinson's Retinal disorders Restenosis Cystic fibrosis Cancer Severe combined immune deficiency (SCID) Hemophilia

Immune response of adenoviral vector

Patients w/o pre-existing immunity can develop immunity Leaky expression of adenoviral proteins - e2/e4 that were not deleted Leads to eventual immune reaction

Components of a retrovirus particle

Pol gene products - viral enzymes - Reverse transcriptase (RT) - Integrase (IN) - Protease (PR) Env gene products - viral envelope (tropism) - Surface subunit (SU) - binds to cellular receptors - Transmembrane subunit (TM) - anchors the protein into the lipid membrane - Surround the virus - lipid bilayer - Glycoproteins in the viral envelope for recognizing specific receptors on host cell + initial infection process Gag gene products - viral core structural proteins - Matrix (MA) - Nucleocapsid (NC) - Capsid (CA)

Retroviruses

RNA virus that inserts copy of its genome into DNA of host cell that it invades - changes genome of that cell Single-stranded positive-sense RNA viruses Infection persists indefinitely

Gene Therapy applications

Replacement of defective genes such as CFTR gene in cystic fibrosis Supplying gene product in large quantities in an unregulated manner - factor IX in hemophilia Introducing genes into cell that do not normally express it - suicide genes in cancer cells Introducing protective genes into target cells such as viral specific ribozymes to treat HIV-1 infections Modulation of immune response such as tolerance induction in autoimmune diseases DNA vaccinations - West Nile Virus DNA vaccine for veterinary applications

Herpes Simplex Type I Virus

Replication deficient virus Developed by eliminating essential regulatory genes Advantage - virus is highly neurotropic - specific Neurotropic virus - tissue specific gene transduction Treat CNS diseases such as Parkinson's Can be selectively depleted by treating w/ ganciclovir - Virus naturally encodes HSV thymidine kinase gene

Why CF is ideal candidate for gene therapy / Requirements for gene therapy to work

Single gene defect Recessive condition (heterozygotes normal, suggesting no gene dosing effects) Main pathology is in the lung which is accessible for treatment Progressive disease w/ virtually normal phenotype at birth, thereby providing a therapeutic window from symptom to treatment Protein replacement therapy in lungs not available and may not be possible for membrane protein requirements for CF gene therapy to work Adequate carrying capacity by gene delivery system Needs to be undetectable by immune system Non-inflammatory Safe to the patients w/ pre-existing lung inflammation Have efficiency sufficient to correct the CF phenotype Have long duration of expression or ability to be safely re-administered

Liposomes

Spherical vesicles composed of synthetic lipid bilayers that mimic the structure of biological membranes Cationic (positively-charged) lipids used for transferring DNA (negatively-charged) into cells Transgene is packaged in vitro w/ the liposomes and used directly for delivering the DNA to a suitable target tissue in vivo Lipid coating protects DNA in vivo and binds to cells - hydrophobic anchor may help go into the cells Packaged DNA enters into cells by direct fusion Efficiency of transfer is relatively low and introduced DNA does not integrate into chromosomal DNA High liposome and DNA concentrations required but both are easy and cheap to manufacture in large amounts

Gene-eluting stents for preventing coronary restenosis

Stent coated w/ PLGA-nanoparticles containing vascular endothelial growth factor (VEGF) gene in animal model system Other potential genes to treat restenosis - PCNA (proliferating cell nuclear antigen), NOS (nitric oxide synthase)

More Sophisticated Retroviral Packaging Systems

To reduce the risk of recombination more sophisticated packaging cell lines were established Based on physical separation of viral genome into different transcriptional units - Different transcriptional units that have minimal homology w/ one another - Commonly use three construct system Replication defective retroviral vector systems - made by separating cis and trans genetic functions of virus into: - Vector construct - contains cis sequences - Helper / packaging plasmids - encode viral proteins

Targeted Gene Therapy using Suicide Gene

Tumor specific promoter attached to prodrug activator gene (herpes simplex thymidine kinase) Normal cell - tumor specific promoter not there so prodrug activator enzyme not transcribed Tumor cell - tumor specific promoter leads to prodrug activator gene being transcribed - Prodrug → toxic metabolite → cell death Gene-directed enzyme-producing therapy (GDEPT) - uses gene taken from cancer cell and then modified with other genes to form enzymes that are harmless to healthy cells Virus-directed enzyme-prodrug therapy - uses virus, such as herpes simplex or cold virus as carrier, or vector, to deliver the modified genes to cancer cells

First human gene therapy trial ADA-SCID

Two girls (ages 4 + 9) w/ ADA SCID were first recipients ADA gene delivered into patient lymphocytes using retroviral vectors Patients continued to receive ADA protein replacement therapy Immune systems restored Sequence Remove ADA-deficient lymphocytes from SCID patient Culture cells in laboratory Infect cells w/ retrovirus that contains normal ADA gene Reinfuse ADA gene corrected lymphocytes back into SCID patient

Biolistic Gene Gun

Uses kinetic energy to deliver nucleic acids inside cells DNA coated on gold or tungsten particles 5-10% transduction efficiency Used for exposed targets such as skin for local transfection Not useful for deep tissues Ex. skeletal myotubes that were hard to transduce w/ viral vectors due to lack of receptors were transfected w/ gene guns

Vectors Associated w/ Gene Therapy for Parkinson's

Using lentiviral vectors w/ three key genes Overexpression of GDNF mediated by lentiviral vector - conferred some protection of the nigrostriatal dopamine terminals against toxicity Ad-GDNF can protect dopaminergic neurons and improve dopamine-dependent behavioral function in young rats w/ progressive 6-OHDA lesions of the nigrostriatal projection AAV-2 (adenoviral) when administered locally transduces only neurons w/i the central nervous system and is particularly efficient in brain regions known to be involved in the pathophysiology of PD - globus pallidus and substantia nigra Strategies in Gene Therapy - Restoration of dopamine synthesis in dorsal striatum - Modulation of activity in basal ganglia downstream of the striatum - Modification of disease progression by neuroprotection

Expanding Host Range for retroviruses

Viruses have limited natural host cell ranges they can infect One may want to limit or expand range of cells susceptible to transduction by gene vector Can replace endogenous envelope proteins of the virus by envelope proteins from other viruses or chimeric proteins Chimera - parts of the viral protein necessary for incorporation into virion as well as sequences meant to interact w/ specific host cell proteins - Viruses in which the envelope proteins have been replaced are pseudotyped viruses Envelope protein often used to pseudotype the retro vector - VSV-G env

General Sequence of Viral Methods

When using viruses, first have to find a way to get desired gene into the virus To get desired gene into viruses - use specifically engineered eukaryotic cells grown in the laboratory Cells are engineered w/ genes (called trans sequences) that express viral proteins - because they supply viral proteins they are called packaging cells If then transfer desired gene linked to correct paackaging sequences (cis) - packaging cells start making viruses carrying desired gene Viruses carrying desired gene - vector viruses Finally vector viruses that carry desired gene can be collected and used to infect target cells from the patient Upon infection, vector virus will deliver desired gene to target cell Procedure Culture packaging cell Make replication incompetent vector virus Deliver gene into target cells of patient by infection

Packaging cell lines + sequence of virus production and infection

also called helper cell lines Immortalized cells grown in the lab transfected w/ DNA encoding for viral gag/pol/env genes gag/pol + env genes are expressed separately in the plasmids - Reduces chance of recombination that can form a replication competent virus Once cell line expressing gag pol and env is established - cells then transfected w/ retroviral vector which contains all the cis acting genes and gene of interest Cells now become factor that produces retroviral vectors Viral vectors produced are replication incompetent b/c trans genes are not included in the vector virus Instead contain designed gene in place of the transgenes Vector virus that is produced can be collected now and is ready to infect any patient target cell - in vivo or ex vivo route When infects target cell - transfers designed gene into chromosome of target cells

Ex Vivo vs. In Vivo Gene Therapy

ex vivo can only be used in targeted cells - removed and then transferred back into the host - cells first removed from human body - cultured in petri dish - can modify cells by transfecting genes into the cells - then select for genetically engineered cells - then amplify cells and return to the body in vivo - gene transfer occurs in patient body

packaging gene of interest

gene of interest can be engineered into vector in context of correct packaging signal and other cis acting sequences if vector was introduced into cell that produces empty viral particles - cell will start to produce viral particles containing therapeutic genes

what happens if psi (packaging signal) is deleted from viral genome

if psi or other cis-acting sequences - viral RNA cannot be packaged into virus results in empty viral particle genetic structure of virus and the existence of proviral form make it easier to manipulate retrovirus to make replication defective vectors for transfer of heterologous gene sequences

germline gene therapy

inheritable genetic modification of germ cells that will pass the selected changes to the next generation - only form of germline therapy has been in transgenic/knockout animals in which exogenous gene is introduced or deleted

somatic gene therapy

not inheritable gene expression only occurs in targeted cells - so far only somatic therapy has been applied to humans

Trans sequences

only have to be present in cell during viral assembly but not necessarily as part of viral genome - gene products can be supplied from genes presented anywhere in genome gag, pol, env gag - viral structural proteins pol - viral enzymatic proteins env - viral envelope proteins

CRISPR

powerful tool for editing genomes to alter gene functions Applications/Advantages - Altering specific loci through insertions, deletions, point mutations, sequence inversions - Treating and preventing the spread of diseases and improving crops - System was recently modified to act as genome regulator by tethering effector domains to Cas9 or guide RNA and as a visualization tool by fusing w/ marker molecules Disadvantages Ethical concerns about germline gene editing - responsible for passing genes onto next generation Safety concerns: - Accuracy - off-field effects, lack of specificity in targeting and incomplete targeting - Once organism is modified they are difficult to distinguish from wt and once released into environment could endanger biodiversity

Simplest First Generation Retroviral Vector System

produces cells known as packaging cell lines contains two DNA constructs w/ significant homology recombination b/w helper (deleted psi) and vector virus (therapeutic gene) could generate replication competent virus

Gene therapy protocols

selecting right gene - which one causes the disease identifying and accessing target cells that require the treatment appropriate gene delivery system proof of principle, safety, efficacy suitable manufacturing and analytical processes

Why expressing multiple genes from a viral vector is desirable

simple retroviral vectors - trans sequences replaced only by gene of choice - limitation is that these can only express one gene multiple genes - can include designed gene + antibiotic resistant gene - allows selection of target cells w/ successful infection - antibiotic resistant gene can serve marker - detection of target cells