Sart Exam 2 - Gene Therapy

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Title: Nude Descending Staircase
Artist: Marcel Duchamp (1912)

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Gene Therapy

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-term describing any procedure to treat of alleviate dz by genetically modifying cells of a patient
-b/c the molecular basis of dz's can vary widely, some gene therapy strategies are particularly suited to certain types of disorder & some to others

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In vivo vs. Ex vivo

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In vivo - genetic material may be transferred directly into cells w/in a patient
Ex vivo - cells may be removed from patient & genetic material inserted into them in vitro, prior to transplanting the modified cells back in patient

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Major Dz Classes Include

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-Infectious Dz
-Cancer
-Inherited Disorders
-Immune System Disorders

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Infectious Diseases

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A result of infection by a virus or bacterial pathogen

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Cancer

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Inappropriate continuation of cell division and cell proliferation as a result of activation of an oncogene or inactivation of a tumor suppressor gene or an apoptosis gene

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Inherited Disorders

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genetic deficiency of an individual gene product or genetically determined inappropriate expression of a gene

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Immune System Disorders

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includes allergies, inflammations, and also autoimmune diseases in which body cells are inappropriately destroyed by immune system cells

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Gene Augmentation Therapy (GAT)

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-for diseases caused by LOSS OF FUNCTION of a gene
-introducing extra copies of the normal gene may increase amount of normal gene product to a level where normal phenotype is restored
-as a result, GAT is targeted at clinical disorders where pathogenesis is reversible

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Targeted Killing of Specific Cells

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-used in cancer gene therapies
-genes directed to target cells & expressed to cause cell killing
-SUICIDE GENE & PRODRUG
-Selectively lytic viruses can be used
-Indirect cell killing uses immunostimulatory genes to provoke/enhance an immune response against target cell

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Suicide Gene

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Inserted genes are expressed to produce a lethal toxin (as cells express toxin gene they die)

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Prodrug

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Gene encoding a Prodrug is inserted conferring susceptibility to killing by a subsequently administered drug (make vulnerable to the drug & specifically target them)

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Immunostimulatory Genes

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make cells express foreign antigen gene or cytokine gene

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Targeted Mutation Correction

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-used when inherited mutation produces a DOMINANT-NEGATIVE EFFECT
-in principle, can be done one at different levels:
-at gene level - by gene targeting methods based
on homologous recombination
-at RNA transcript level - by using particular types
of therapeutic ribozymes or therapeutic RNA
editing

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Targeted Inhibition of Gene Expression

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-Dz cells display novel gene product or inappropriate expression of a gene
-many cancer cases, infectious dz's
-used to specifically block expression of single gene at DNA, RNA, or protein levels
-allele-specific inhibition of expression may be possible in some cases, permitting therapies for some disorders resulting from dominant negative effects

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Anti-sense Genes

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A gene that when expressed, will make nothing

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Somatic V.s. Germline

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-Current gene therapy = somatic gene therapy: intro of genes into somatic cells of affected individual
-Prospect of human germline gene therapy raises number of ethical concerns & is not sanctioned

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Technology of Classical Gene Therapy

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-Genes can be inserted into cells of patients by DIRECT & INDIRECT routes & the inserted genes can integrate into chromosomes or remain extra-chromosomal
-Ex vivo gene transfer
-In vivo gene transfer

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Ex Vivo Gene Transfer

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-Transfer of clones genes into cells grown in culture
-cells successfully transformed selected, expanded by cell culture in vitro, reintroduced to patient
-Autologous cells used
-cells collected initially from patient to be treated & grown in culture before being reintroduced to same individual
-occasionally, cells implanted are allogeneic

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Autologous Cells

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-cells from the same patient
-used to avoid immune system rejection of the introduced cells

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Allogeneic Cells

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-cells from someone else, who is an HLA match
-HLA matching important to avoid immune rejection

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In Vivo Gene Transfer

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-Genetic modification of the cells of a patient in situ
-Here the cloned genes are transferred directly into the tissues of the patient
-Useful in tissues where individual cells cannot be cultured in vitro in sufficient numbers and/or where cultured cells cannot be re-implanted efficiently in patients

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Principles of Gene Transfer

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-classical gene therapies normally require efficient transfer of cloned genes into dz cells so that the introduced genes are expressed at suitable high levels
-size of DNA fragments transferrable are limited
-Artificial minigene may be used: cDNA sequence containing complete coding DNA sequence
-Inserted genes may integrate into chromosomes of cell, or remain as extrachromosomal genetic elements (episomes)

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Advantages of Gene Integration into Chromosomes

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-Gene can be perpetuated by chromosomal replication following cell division
-long-term stable expression may be obtained
-possibility of a cure for some disorders
-Key cells to target are stem cells
-immortal pop. of cells from which all other cells of
the tissue are derived
-high efficiency gene transfer into stem cells, and
subsequent stable high level expression of a
suitable introduced gene; can provides possibility
of curing a genetic disorder

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Disadvantages of Chromosomal Integration

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-Insertion occurs almost randomly
-Location of inserted genes can vary enormously from cell to cell
-Inserted genes may not be expressed
-Integration event can result in death of host cell
-insertion may occur w/in crucially imp. gene,
which could inactivate it
-could cause cancer
-integration could disturb normal expression patterns of genes that control cell division or cell proliferation

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Non-Integrated Genes

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-insert genes into cells where they remain as extra-chromosomal elements and may be expressed at high levels
-Disadvantages: cells actively dividing, introduced gene may not segregate equally to daughter cells - long term expression may be a problem
-repeated treatments involve gene transfer necessary
-there many be no need for stable long-term expression

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No Need For Stable Long-Term Expression

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-Cancer gene therapies
-Expression of genes into cancers cells - malignancy has been eliminated - the therapeutic gene may no longer be needed

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Vectors

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-mammalian viral vectors have high efficiency of gene transfer
-method depends on nature of target tissue & whether transfer is to cultured cells ex vivo or to cells of patient in vivo
-no one gene transfer system is ideal
-mammalian virus vectors preferred vehicle for gene transfer b/c of high efficiency of transduction into human cells

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Oncoretroviral Vectors

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-retroviruses are RNA viruses which possess a reverse transcriptase function, enabling them to synthesize a complimentary DNA form
-viral RNA genome is transcribed & integrates the resulting DNA copy into a single site in host cell chromosomes
-very efficient at transferring DNA into cells
-integrated DNA can be stably propagated

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Adenovirus Vectors

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-DNA viruses produce infections of upper respiratory tract
-Human viruses can be produced at very high titers in culture & are able to infect large #'s of diff. human cell types including non-dividing cells
-Entry into cells occurs by receptor-mediated endocytosis & transduction efficiency is very high
-Large viruses have potential for accepting large inserts
-Adenoviruses enter cells by receptor-mediated endocytosis

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Adenovirus Disadvantages

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-Expression is short-lived: inserted DNA does not integrate, expression of inserted genes can be sustained over short periods only
-Non-target cells at risk: b/c they can infect virtually all human cells, pose a risk in some therapies that are designed to kill cancer cells w/out causing toxicity to normal surrounding cells
-Risk of immune response: can generate unwanted immune responses, causing chronic inflammation

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Adeno-associated Virus Vectors (AAVs)

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-group of small, single-stranded DNA viruses which cannot usually undergo productive infection w/out co-infection by a helper virus
-adenovirus genes have been deleted from some newer adenovirus vectors ('gutless vectors')
-provide high degree of safety: b/c 96% of parental AAV genome has been deleted, AAV vectors only contain the gene of interest

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Herpes Simplex Virus Vectors

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-HSV vectors are tropic for central nervous system (CNS) & can establish lifelong latent infections in neurons
-Have comparatively large insert size capacity (>20kb)
-Non-integrating & long-term expression of transferred genes is not possible
-Major app. expected to be in delivering genes into neurons for treatment of neurological dz's (Parkinson's, & treating CNS tumors)

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Lentiviruses

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-Complex retroviruses that infect macrophages & lymphocytes (ex: HIV)
-Able to transduce non-dividing cells
-Able to integrate into chromosome
-In HIV, preintegration complex contains nuclear localization signals that permit its active transport through nuclear pores into nucleus during interphase

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Nonviral Vector Systems for Gene Therapy

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-Liposomes
-Direct injection/particle bombardment
-Receptor-mediated endocytosis

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Liposomes

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-Spherical vesicles composed of synthetic lipid bilayers, mimic structure of biological membranes
-Transferred DNA packaged into vitro w/liposomes & used directly for transferring DNA to suitable target tissue in vivo
-Lipid coating allows DNA to survive in vivo, bind to cells & be endocytosed into cells
-Cationic liposomes - popular vehicles for gene transfer in in vivo gene therapy
-Unlike viral vectors, DNA/lipid complexes are easy to prepare & there's no limit to size of DNA that's transferred
-Efficiency of gene transfer low, introduced DNA not designed to integrate into chromosomal DNA

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Direct Injection/Particle Bombardment

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-"gene gun"
-DNA injected directly w/syringe & needle into spec. tissue (ex: muscle)
-Alt. approach uses particle bombardment techniques: DNA coated on metal pellets & fired from special gun into cells
-Simple & comparatively safe
-Poor efficiency of gene transfer & low level of stable integration of injected DNA
-May be less of a problem in tissues which don't regularly proliferate - DNA may continue to be expressed for several months

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Receptor-Mediated Endocytosis

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-DNA coupled to targeting molecule - can bind to spec. cell surface receptor - transfer DNA into cells via endocytosis
-Hepatocytes
-General approach: utilizes transferring receptor: expressed in many cells types (ex: proliferating & hemopoietic cells)
-Gene transfer efficiency high
-Does not allow integration of transferred genes
-Protein: DNA complexes not particularly stable in serum
-DNA conjugates may be entrapped in endosomes & degraded in lysosomes

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Gene Therapy for Inherited Disorders

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-Single gene disorders
-Recessively inherited disorders

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Single Gene Disorders

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-individual severely affected & where there's no effective treatment, more obvious candidates for gene therapy
-w/in single gene disorder category, but differing pathogenesis means certain single gene disorders will be more amenable to gene therapy approaches than others

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Recessively Inherited Disorders

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-easiest inherited disorders to treat by gene therapy
-dz results from simple deficiency of specific gene product generally most amenable to treatment: high level expression of introduced normal allele should be sufficient to overcome genetic deficiency
-mutations almost always simple loss-of-function mutations
-affected ind. have deficient expression from both alleles & so dz phenotype is due to complete or almost complete absence of normal gene expression
-Heterozygotes have ~50% of normal gene product & are normally asymptomatic
-amenable to gene augmentation therapy

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ADA Definiciency

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-first gene therapy trial for inherited disorder in 1990
-Ashanthi DeSilva - 4yrs old w/adenosine deaminase deficiency
-ADA involved in purine salvage pathway of nucleic acid degradation & is housekeeping enzyme - synthesized in many diff. types of cells
-Severe consequences in case of T-lymphocytes, one of major classes of immune system cells
-ADA deficiency good candidate for gene therapy
-ADA gene small, previously cloned & studied
-Target cells = T-cells & easily accessible & easy to culture, enabling ex vivo gene therapy
-Disorder is recessively inherited & gene expression not tightly controlled
-Transfer of normal ADA genes into ADA-T cells noted to result in restoration of normal phenotype

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ADA Gene Therapy

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-cloning normal ADA gene into retroviral vector
-transfecting ADA recombinant into cultured ADA-T lymphocytes from patient
-identifying resulting ADA+ T cells & expanding them in culture
-re-implanting these cells in patient

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Gene Therapy for Neoplastic Disorders: Cancer Gene Therapies

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-Diff. approaches used for cancer gene therapy
-Treatments based on targeted killing of dz cells, by introducing genes that encode toxins or by provoking enhanced immune responses
-Targeting single genes, such as TP53 gene augmentation therapy
-Delivery of antisense KRAS genes in case of some forms of non-small-cell lung cancer

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Gene Therapy for Neoplastic Disorders: Prodrug Gene Example

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-Brain tumor cells, glioblastoma multiforme
-retrovirus-mediated transfer of gene encoding a prodrug- a reagent that confers sensitivity to cell killing following subsequent administration of suitable drug
-retroviruses provided in form of murine fibroblasts are producing retroviral vectors (retroviral vector-producing cells or VPCs)
-cells directly implanted into multiple areas w/in growing tumors
-once injected, VPCs continuously produce retroviral particles w/in tumor mass, transferring genes into surrounding tumor cells
-retroviral vectors produced by cells used to transfer gene encoding a prodrug, herpes simplex thymidine kinase (HSV-tk) into tumor cells
-this reagent confer sensitivity to drug gancyclvir

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Gancylclovir

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-b/c retroviruses infect only dividing cells, they infect tumor cells, but not normal differentiated brain cells
-implanted VPCs transferred the HSV-tk gene to neighboring tumor cells, rendering them susceptible to killing following subsequent intravenous administration of gancyclovir

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Gene Therapy for Infectious Disorders

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-strategies can involve provoking a specific immune response or spec. killing of infected cells
-increasingly pop. additional approach targets life-cycle of infectious agent, reducing its ability to undergo productive infection
-some infectious agents may be undergoing rapid evolution- present problems for any general therapy (classic ex: is AIDS, infectious agent, HIV-1, mutates rapidly)
-Interfering w/ life-cycle of infectious agent - 3 strategies

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Interfering w/ life-cycle of infectious agent: 3 strategies

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-Blocking HIV-1 infection - ex: block T-cells by using soluble CD4 'decoys'
-Inhibition at the RNA level - TAR & RRE
-Inhibition at the protein level - introducing genes that encode for dominant negative