Biotechnology – Gene Therapy – Flashcards

-'Treatment that consists of introducing into a patient a normal copy of one or more defective genes responsible for the patient's disease' -classical gene therapy ->1964: Tatum, Lederberg, Kornberg suggested that the future of genetic disease therapy would be curing disorders by replacing defective genes with functional ones. -->cystic fibrosis, muscular dystrophy, multiple sclerosis. ---> but the required tools did not exist -Can be somatic or germ line -only somatic therapy has been done to date ->genetic alterations wont be passed to future generations. -Germ-line therapy has potential to completely cue a genetic disorder ->if defect corrected in sperm or egg->all progeny is modif. -->ethical implications ->illegal in many countries.

-Preferred method as easier to transform cell in vitro rather than in vivo. -Cells are removed from the body & cultured in vitro. -Cultured cells are transformed with therapeutic gene. -Population expanded -> transformed cells are selected for by antibiotic resistance. -Cells are then returned to the patient->transgene expression & effect monitered. -usually blood cells as they are the easiest to remove & return but can also use bone marrow.

-Genetic material is transferred directly into the body. -Used for cells that cannot be effectively returned back to the body. -Less control over the levels of expression, DNA integration, possible immune response against delivery agents -> less control once virus is injected.

-Non integrated ->Episomes: essentially plasmids (circular-> inhibits degradation by cellular exonucleases (nibble free ends)) ->Transient expression: can be quite stable but lost when cell divides -> repeated treatments required. -Integrated (part of bacterial DNA - stable & retained in dividing cells) ->Random -->In heterochromatin (tight) can be inactivated -->In euchromatin (loose) can disrupt important genes or activate oncogenes --> Targeted -> ideal but difficult.

-Gene Transplantation: to patient with gene deletion -> patient's immune system may reject it as it has never produced this protein before -Gene Augmentation: introducing functional gene, mutant copy still remains. -Gene Correction: replacing a mutated gene with functional copy -'Suicide' Gene Therapy: targeting specific cells with lethal gene -Gene Ablation: targeted inhibtion of gene expression.

-Any well-studied monogenic (many genes contribute to the disease) disorder with specific. easily identifiable & well-studied disease alleles. ->Haemophilia A - absence of clotting factor VIII ->Haemophilia B - absence of clotting factor IX ->cystic fibrosis - defective chloride channel protein ->Muscular dystrophy - defective muscle protein (dystophin) ->sickle-cell anaemia - defective beta globin -> severe combined immunodeficiency disorder (SCID) - any one of several genes fail to make a protein essential for T and B cell function

- patients lack cell-mediated immune responses (B, T, NK cells) & antibodies. - number of genetic defects can give rise to disorder ->(25%): defective adenosine deaminase (ADA) --> lacks ability to recycle purines, toxic intermediates accumulate -->deleterious to rapidly developing immune system -Treatment: => Bone marrow transplant- requires histocompatible match->even siblings differ -->No immune system so can not reject the transplant -> can still cause graft-vs-host disease- new immune system's T cells attacks the existing immune system. ->donated marrow could contain latent virus --> attack existing i.s before the new i.s is formed -(healthy individuals i.s keeps the latent virus in check) =>ADA enzyme therapy -ADA is purified from cows & conjugated with polyethylene glycerol (PEG) to delay breakdown in blood. -expensive - $500,000 p.a -needs to be constantly reinjected -can develop immune response & resistance to therapeutic enzyme. -> if you can make 10% of ADA-> its enough. =>Raise child in sterile environment - keep alive till cure -> David 'bubble boy'- X-linked form of SIDS (DNA) -growing psychological instable so attempted bone marrow transplant from sister -> was successful but contained the Epstein-Barr virus-> died. => Gene Therapy -ideal candiate for first gene therapy as: -monogenic (caused by mutation in 1 gene), genetic basis well characterised, gene cloned --lethal so for some patients it is there only option --blood/marrow cells allow ex vivo therapy to occur --variable gene expression levels are well tolerated ->just need to get gene into the cell so it can be expressed at some level

-original proposal was to use human bone marrow stem cells & do an ex vivo transfection -> provide permanent correction. (W. French Anderson) -> not possible to get good transfection of stem cells with the early retrovirses vectors that were avaliable. -T-cells were suggested as a target (Michael Blaese) ->T-cells do not live very long unless stim. by antigen. ->once stim.->long lived, rapidly dividing 'memory' T-cells ->i.s response is governed by previously-generated memory cells -> hoping for 'snowballing' effect.

-Recombinant DNA Advisory Committee (RAC) & FDA took almost 2 years to apprive trial with requirements -> insisted on non-replication vector ->PEG-ADA treatment to continue throughout ->entensive pre-clinical data required -->retrovirus couldnt replicate -->ADA successfully delievered & expressed in Ashanti's culutred T cells -->recombinant cells not subject to toxic buildup of deoxyadenosine -->equivalent experiments successful & safe in animal models (mouse & then monkey)

-Ashanti de Silva, 4 year old ADA-deficient SCID patient ->T cells are purified from blood & grown in tissue culture -stimulated to proliferate with IL2 (a growth stimulating cytokine) -infected with disabled retrovirus carrying functional ADA gene -virus cannot replicate, only integrates into genome -no selection -> relies on large excess of virus. => worked! -T-cells are now expressing functional ADA -4 infusions over 4 months & after 6 months T-cell levels are rising. -serum ADA levels are 1/4 with near normal T cell count with 20-25% transgenic. -given continual PEG-ADA levels as unethical to stop ->unable to see whether T-cells that make ADA will out compete those that don't. -> could be countering effectiveness of the gene therapy as it allows crippled non-recombinants T cells to compete better ->preventing a long term more permenant cure -unclear the effect this is having as T-cells only live 6-12 months in blood so infusions are required

-look at a normal retroviral life cycle -Minimal retrovirus structure: ->outer envelope- derived from plasma membrane of host ->envelope proteins embedded in this membrane --> virally encoded, target specific host cell receptors --> give specificity for host & preference for cell-type ->capsid (structural protein shell around the RNA genome & reverse transcriptase) ->single-stranded RNA genome ->RNA associated reverse transcriptase/integrase enzymes -->carried with virus to copy ssRNA to dsDNA & then integrate the dsDNA into the host genome. -> want to get rid of parts that are going to destroy host cells.

-LTR - Long Terminal Repeats; dual function -PSI (looks like a trident) -'packaging sequence' - recognized by gag proteins for viral assembly. -gag- 'group antigens' (capsid) -> proteins are have antigens to shield & help integration into other cells -pol - polymerase (reverse transcriptase/integrase) -env- envelope proteins -LTR- Long Terminal Repeats; dual function -> repeats recognised by integrase -> dsDNA integration into host genome ->contains enhancer sequences give high levels of transcription of enclosed genes

-LTR-> for integration & expression -PSI->for packaging of a virus -> where construct is expressed -Therapeutic Gene->want to turn into dsDNA & integrate into genome -LTR ->no gag, pol, env (all genes necessary for replication) ->cannot replicate once integrated -> will just express the therapeutic gene -use a 'package' strain ->has gag,pol,env genes in chromosome, but no psi or LTRS so these genes themselves can not be packaged

-used same vector but targeted stem cells, not T-cells ->Donald Kohn, paediatrician colleague of Blaese, diagnosed 3 in utero cases of ACA-SCID (early 1993) ->obtained permission for gene therapy trial --> got blood stem cells from umbilical cords ->methods for stem cell enrichment & division had been developed-> feasible to transfect with retrovirus -injected transfected cells into marrow, monitored T cell expression of ADA -Qualified success -> only very low proportion of injected cells 'took' in marrow ->0.01-0.1% of T-cells expressing transgene ->got permission to wean patients off PEG-ADA ->as PEG-ADA reduced, percentage of T cells expressing transgene rose 10-100 fold -patients still dependent on PEG-ADA but trial important in two respects ->showed that stem cell engineering was possible ->validated previous hypothesis that PEG-ADA was promoting surivial of untransformed cells.

-insert semi-randomly into the host genome ->integration mechanism targets expressed genes (euchromatin (open) over heterochromatin (closed)) ->high percentage of integration events will disrupt genes ->small proportion may activate silenced genes; but also potential for oncogene activation, tumour development. -->enhancer seq. in LTR made this worse. -Large trial in France, 1997-2002, 11 boys with X-linked SCID -> all now have adaptive immune system ->lack gammaC subunit for interleukin receptor -> cant make T-cells ->gene well-characterised, cloned -Took bone marrow, selected & cultured stem cells, transfected & returned to patients ->previous trals had first destroyed a large proportion of existing marrow cells to 'make room' for transplanted cells -> 3 of 11 boys developed acute T-cell leukemia -> tumors derived from single T cell where viral integration had activated an oncogene.-> one dead

-Sept 1999 -> died in gene therapy trial -Ornithine transcarbamylase deficiency trial ->patients with impaired in urea cycle -> NH3 (ammounia) buildup -Mild form can be treated with sodium benzoate -Severe form is fatal in babies -Gelsinger had a mild form -> mosaic -> mutation occurred at 4 cell embryo -> 75% cells were healthy -volunteered for Phase 1 trial-> testing safety/dosage of OTC-carrying adenovirus -> usually done on healthy subjects. -died of immune hyper-response to large viral load directly injected into blood. ->first to die of gene therapy & not disease ->led to serious q. over clinical practices -->other patients had shown allergic reponses at lower doses -->Gelsinger had high levels of NH3 from his disorder - was he fit to be on the trial?

-should clinicians test directly in affected babies, or should first conduct Phase 1 safety trials in healthy adults? ->what about partially affected adults like Jesse? ->if not babies- what about the ones that die in the meantime -if afflicted individuals are used in Phase I trials for safety- how to determine whether adverse affects were caused by disease or therapy? ->If patients included on "compassionate grounds", should deaths be counted against drug? ->How about success stories? ->Who should pay for compassionate trials? -Placebos- is it fair to include patients who are not receiving the real therapy for comparison? ->If not, how to tell if the treated group were better (or worse) off? ->If there appears to be a clear difference early in the trial, is it ethical to keep administering placebos? ->Is it ethical to allow some patients to die to prove the point & make it easier to save others later on?

1. treatment of a life-threatening disorder 2. preclinical data sufficiently compelling to offer a genuine prospect of cure 3. an absence of any reasonable therapeutic alternatives -could justify patient inclusion in a gene therapy trial -rebirth has taken form of new & promising vectors, & ongoing development of anti-cancer gene therapy

-Easy to manipulate (easy to insert transgene,size tolerated) -Precise (safe), stable, expressed introduction of transgene -should not elicit immune response from host -usually want it to be disabled (non-replicatng) -wide host range (can test in animal models) -may be desirable to target specific cell types

->MoMLV -8000 bp- (>6000bp-> need a diff. retrovirus) Pros: -Integrate into genome - long term expression of transgene -typically have wide host range Cons: -Small capacity for therapeutic genes -Infectivity limited to dividing cells -Large scale produciton expensive & technically difficult -integration is random with potential for oncogensis

-dsDNA viruses (cold virus) (40,000-50,000bp) Pros: -Tolerate larger transgene inserts -very infectious with high levels of gene expression -non-integrative: low potential for oncognesis -can infect dividing & non-dividing cells Cons: -Non-integrative: expression is transient -Native coat proteins can elicit strong immune response ->everyone has had a cold! (biggest drawback) -->can be addressed by GE by modifying coat proteins ->use same packaging strain to make non-replicating (gutted) virus

-ssDNA ->can not replicate on its own -only 2 ORFs in the genome ->needs adenovirus or HSV co-infection to provide replicative functions -in absence of other virus- DNA integrates at precise location on chromosome 19 -elicits no immune response ->doesnt cause disease Pros: -Not associated with disease ->Virtually no immune response -Infects both dividing & non-dividing cells -Integrates DNA at precise & safe location in genome Cons: -small (can only contain up to 4.5kb of DNA) ->vector of choice for large number of ongoing trials

-like biological membranes & lipid bilayers -typically polar head group will be cationic (positively charged) -> in soln. will form vesicles around anionic DNA (has negative charge) ->can merge with cell membranes (releasing DNA directly into the cytoplasm) ->can also modify surfaces with antibodies to target specific cell receptors (receptors over-represented in tumors) Pros: -easy to produce -biologically inert (no immune response) -non-integrating so no cancer risk Cons: -poor delivery -majority of DNA degraded before reaching nucleus -non-integrating so expression transient

-Has been trialled for Duchenne Muscular Dystrophy (DMD) ->X-linked recessive disorder (affects 1:4000 boys) -Massive gene (2.3 Mb) -> too large viral delivery -cheap & easy to administer but inefficient

-Next generation is the use of lentiviruses eg HIV --have the ability to infect non-dividing cells