Final - Gene therapy, RNAi and Aptamers

viruses (retroviruses & adenoviruses), non-viral carriers (plasmid-containing liposomes/lipoplexes

mechanism partly understood with retroviral vectors, mechanism of lipsome vectors or naked DNA less well understood

1. in vitro approach (most common, remove target cells from body, cultured in vitro & incubated with vector containing nucleic acid, altered cells reintroduced into pt's body); 2. direct injection/administration (nucleic-acid-containing vector into target cell); 3. alternative (in vivo) approach (method of choice) (development of vectors that recognize and bind to specific predefined cell types, administered by IV injection, only deliver nucleic acid to specified target cell, *no such system has been developed)

introduction of cystic fibrosis transmembrane conductance regulator (CFTR) gene into airway epithelial cells of systic fibrosis pts

introduction of a novel gene into white blood cells whose protein product is capable of in some way interfering with HIV replication

desired duration of subsequent expression of gene product; regulation of expression of transferred gene; choice of target cells

24% of all gene therapy trials use retroviral vectors; enveloped viruses; genome consists of ssRNA; upon entry into cells, RNA is reverse transcribed & yields dsDNA; this integrates into host cell genome

3 basic structural genes: gag (cosed for core viral proteins), pol (codes for reverse transcriptase) & env (codes for viral envelope protein); at either end of viral genome are long terminal repeats that keep promoter & enhancer regions required to promote integration into host DNA; immediately adjacent to 5'LTR is packing sequence (si region, required to promote viral RNA packaging)

1. ability to effectively enter various cell types; 2. ability to integrate their genome into host cell genome in a stable, long-term fashion

replace endogenous viral genes for normal replication with exogenous gene; remove viral structural genes (means vector cannot replicate itself); genetic material introduced into 'packing cell' (recombinant cells engineered to contain gag, pol, env structural genes; packing cells produce mature, but replication-deficient, viral particles, viral particles function as so-called one-time gene transfer systems)

inclusion of 5' end of gag gene (enhances levels of vector production by up to 200-fold); specific promoters introduced to attempt to control expression of inserted gene (use of tissue-specific promoters to limit expression of desired gene to a specific tissue)

derived from Maloney murine leukemia virus (MoMuL V)

retroviruses have been studied in detail (biochemistry & molecular biology are well understood); efficiency of gene transfer to most sensitive cell types is very high; integrated DNA can be subject to long-term relatively high-level expression retroviruses are promiscuous, in that they infect a variety of dividing cells (good if we can control; bad if we can't); complete copies of proviral DNA passed to daughter cells if recipient cell divides; good, high-level titer stocks of replication-incompetent retroviral particles produced; safety studies using retroviral vectors have been carried out on various animal species

they are relatively labile (easy to propagate, but often damaged by subsequent purification and concentration); ability to infect only dividing cells restricts their use; they are promiscuous in that they infect a variety of dividing cells; they ingegrate randomly into chromosomes of recipient cells (integration into a gene with a critical role could damage cellular function, disruption of a tumor suppressor gene could give rise to cellular transformation, integration adjacent to quiescent cellular proto-oncogenes could result in activation)

2 children who had received retroviral-based gene therapy 2 years previously developed a leukaemic-like condition; initial therapy aimed to treat SCID-X1, and worked in 9 of 10 patients, but the 2 youngest pts developed uncontrolled proliferation of mature t-lymphocytes 30 and 34 months after therapy

relatively large, non-enveloped structures, housing dsDNA; genome is much larger and more complex than retroviruses; usually only a small fraction of genome is removed when using as a vector; upon cellular infection, adenoviral DNA becomes localized in nucleus, but *does not integrate into host cell DNA*

ability to infect non-dividing cells efficiently; usually observed expression of large quantities of desired gene products

failure to integrate into host cell (survival & duration of action is limited, theoretically could do repeat administrations [but prompt strong immune response])

small, ssDNA virus; genome consists of only 2 genes; cannot replicate autonomously; can only replicate in presence of co-infecting adenovirus; not associated with any known diseaese; is a mechanism of small gene transfer into nondividing cells; facilitates long-term expression; appears to be integrated into recipient cell genome

particularly useful in delivering genes to neurons of nervous system (latent in nondividing cells [genome remains in unintegrated form], proven difficult to generate replication-incompetent yet viable herpes simplex particles [some still damage/destroy cells]; suitable and safe vector variations of herpes simplex virus not available yet)

member of alphavirus family with ssRNA; can infect broad range of both insect and vertebrate cells; mature virion particles consist of RNA genome complexed with capsid protein C [enveloped by lipid bilayer with 2 additional viral proteins {E1 & E2} embedded, E2 polypeptide mediates viral binding to surface receptors of susceptible cells, major mammalian cell surface receptor is widely distributed laminin recpetor); simple, robust and capable of infecting non-dividing cells; generally supports high levels of gene expression; broad host range so lacks inherent targeting specificity characteristics

displays altered host cell specificity (nucleotide sequence coding for IgG bingin domain of Staphylococcus aureus [protein A], inserted into E2 viral gene [disrupts lamnin binding], protein A domain allows chimeric E2 product to bind monoclonal antibodies [IgG], potentially capable of being specifically targeted to any desired cell type), initial studies using this system have proved encouraging (altered virus [without monoclonal antibody] failed to infect wide variety of cell lines, incubating with monoclonal antibody transduced cells expressing CD4, CD33, and HLA), full potential of this approach will also require more detailed characterization

column chromatography employed on industrial scale: intact cells/cellular debris is removed by filtration, physical degradation of DNA by addition of nuclease enzyme to decrease viscosity, solvent/detergent treatment step in order to inactivate any enveloped contaminant viruses, high-resolution purification achieved ion-exchange and gel-filtration chromatography, product concentrations steps by ultrafiltration, final product is then filter sterilized and filled into glass vials, purified vectors stored either refrigerated or frozen (shelf-life of 2+ years)

low/non-immunogenic, non-occurrence of integration of therapeutic gene into host chromosome (eliminates potential to disrupt essential host genes or to activate host oncogenes)

IM injection into mouse muscle (beta-galactosidase gene as reporter), activity could persist from a few months to life time, transfection rate was low (1-2 % of muscle fibers assimilated DNA), DNA was not integrated into host cells' chromosomes, showed naked DNA injected could be spontaneously taken up and expressed

DNA coated on microscopic godl beads, porpelled into epidermis of test animals and is expressed in animal's skin cells, resulted in effective immunization of animal against influenza, similar results with other pathogens, assumed expressed antigen is secreted by cell and exposed to immune surveillance

rarely achieves meaningful cell transfection, due to high nuclease levels present in serum (low nuclease activity in muscle)

Gene of interest (with appropriate promoters in circular plasmid), additional molecules include various lipids or some polypeptides (generally have a + charge, interact with - charged DNA molecules, function is to stabilize DNA, protect DNA from serum nucleases, help target DNA to particular cell types or away from other cell types

cationic lipids and polylysine chains (most common) (aggregate in aqueous-based systems, form vesicles/liposomes, form complex 3D lattice-like system [physical protection to therapeutic gene]); gene therapy results with cationic lipid lipoplexes (mixed effects) (process of lipoplex formation is not easily controlled, identical conditions does not equal structurally identical product, in vitro test results does not equal in vivo performance); polylysine-based systems (positive charge forms electrostatic complexes with DNA, stability of such 'polyplexes' in biological fluids is problem, polyplexes tend to be rapidly removed from circulation [attachment of PEG molecule helps])

blood-related issues, biodistribution profile, cellular targeting (overall + charge [non-specific interactions with cells & serum proteins]), following IV injection complexes accumulate in lung & liver, targeting specific cell types poses unmet technical challenge (incorporation of antibodies directed against specific cell surface antigens, believed that ionic interactions constitute predominant binding force), most effective means of delivery - inject into/beside target area

cellular entry achieved via endocytosis (proportion of endocytosed plasmid DNA escapes from endosome, enters cytoplasm, thereby escaping liposomal destruction, anionic lipids may fuse directly with endosomal membranes, generally DNA is released in free form

incorporation of selected hydrophobic peptides (many viruses naturally enter animal cells via receptor-mediated endocytosis, viruses have evolved efficient means of endosomal escape [membrane disrupting peptides derived from viral coat proteins]), in cytoplasm, a proportion of plasmid molecules degraded by cytoplasmic nucleases, 2 potential routes by which plasmid DNA could reach nucleus: direct nuclear entry as consequence of nuclear membrane breakdown (mitosis) and transport through nuclear pores (passive diffusion or active transport), estimated that one in 10^4 or 10^5 plasmids taken up by endocytosis is expressed

Fermentation (upstream processing)(promotes microbial cell replication [biosynthesis of large quantities of plasmid]); harvested (collected)(centrifugation or microfiltration); resuspension and disruption (release the plasmids) (addition of lysis reagent consisting of NaOH [pH] and SDS [impurities: cell wall decris and some intact cells, proteins, genomic DNA, RNA, low molevular mass metabolites, endotoxin]); high-salt neutralization (potassium acetate) (promotes formation of aggregates of genomic DNA and SDS-protein complexes, removed by centrifugation or filtration); plasmid precipitation (isopropanol or ethanol, resuspended); chromatographic purification (gel-filtration, ion exhcnage, reverse-phase and affinity systems, capture chormatography [plasmids binding to chromatographic beads] low plasmid-binding capacities observed); analytical techniques (contaminating nucleic acid/proteins, endotoxin and sterility tests); formulation (problem of aggregation [aqueous suspensions of these], possible solution - flash freezing, cryoprotectants [glucose, sucrose, trehalose], freeze-dried product)

small number of genes responsible have been identified and studied (completion of human genome project should accelerate); none of 1st-gen. gene-delivering vectors have proven fully satisfactory; some genetic diseases are quite complex with several organs/cell types being affected; refulation of expression levels of genes transferred has proven problematic; drug companies interest in prevalent diseases

not only dependent on presece of alpha- and beta-globin genes of correct sequence; also dependent upon detailed regulation of gene expression; regulation of expression of transferred genes is beyond capability of gene therapy

lack adenosine deaminase (ADA) activity - degradation of purine nucleosides [catalyses removal of ammonia from adenosine, forming inosine, leads to T- and B-lymphocyte dysfunction, SCID sufferers must be kept in an essentially sterile environment], Gene therapy trials initiated in 1990 [isolation of peripheral lymphocytes, in vitro introduction of human ADA gene into these cells using retroviral vector, expansion by culture in vitro, treated cells re-injected into patient, therapy repeated every 6-8 weeks); approach appeared successful

immortal, outine transfuction of hematopoietic stem cells has proven technically difficult (found only in low quantities in bone marrow, lack of suitable assay for stem cells, revent progess has been made [routine transduction will likely be achievable withiin next few years])

absence or presence of defective form of liver LDL receptors, results in highly elevated serum cholesterol levels, accompanied by early onset of serious vascular disease, approach: initial removal of relatively large portion of liver, hepatocytes derived from liver are then cultured in vitro, gene transfer undertaken using retoroviral vectors, corrected hepatocytes are infused back into liver via catheter, trasduction of only a small proportion of hepatocytes is normally observed, expression of corrective gene can also be variable, in vivo approaches using both viral and non-viral approaches also currently bveing assessed)

cf gene: identifies in 1989, codes for CFTR (170 kDa protein that serves as chloride channel in epithelial cells), inderitance of mutant cftr gene from both parents results in cystic fibrosis phenotype (most severely affected sites are respiratory epithelial cells, focus of attempts at correttive gene therapy); Most inherited monogenetic disease in Europe and US (typical life expectancy of 40 years); over 1/3 of 100 or so gene therapy trias specifially targeted cystic fibrosis; several vectors have been used (most notable systems include adenoviruses and cationic lipsomes, vector delivery to target cells can be achieved directly be aerosol technology, CFTR cDNA to airway epithelial cells demonstrated with use of both vector types, 5-10% of target cell population must receive and express CFTR gene [thislevel integration has not been achieved so far, gene expression has often been transient)

requirement for improved, more target-specific vector systems; requirement for better understanding of how cancer cells evade normal immune response; for ethical reasons (most patients treated suffering from advanced & wide spread terminal cancer, earlier stages of cancer will probably prove to be more sponsive to gene therapy)

introduction of TNF gene into TIL: reintroduced TIL cells reintroduced into body synthesize TNF at tumor site, approach has since been broadened (introducing genes coding for a range of immunostimulatory cytokines [IL-2, IL-4, IFN-y and GM-CSF into TILs] variation of this approach involves introduction of cytokine genes into tumor cells), desired result is killing umore cells & vaccinating patient, strategy: removal of target cells from body, culture in vitro, introduction of desired gene (mainly using retroviral vectors), reintroduction of altered cells into body

insertion of a copy of a tumore suppressor gene into cancer cell (deficiency in p53 implicated in development of various human cancers, insertion of p53 gene in some p53-deficient tumor cell lines induces death, potential weakness is that 100% would have to be succcessfully treated for a cure [combination with conventional approaches required, sole gene-therapy-based medicine approved {in China only} uses this approach]); Introduction of a 'sensitivity' gene into tumor cells (gene product with ability to convert a non-toxic-pro-drug into toxic substance [within cells leading to their selective destruction], model system most used to appraise such an approach [thymidine kinasse gene of herpes simplex virus])

Often expressed by cancer cells resistant to chemotherapy; pumps out a range of chemotherapeutic drugs (daunorubicin, taxol, vinblastine, vincistine and others); animal studies confirmed MDR-1ction into stem cells protects these cells (large doses of taxol could be used); being appraised for breast and ovarian cancer and brain tumors

Dysfunctional gag, tat, or env into viral sensitive cells: mutant forms of gag shown to be capable of inhibiting viral replication, interferencce with correct assembly in infected cells; Introductino of gene for antibody binding evelope proteins: may also interefere with viral assembly in infected cells

HIV cell surface receptor, would bind cirulating virions (block their ability to 'dock' at sensitive cells), proved to be the case in vitro but not in early in vivo studies

Costs: likely broadly similar to cost of present-day biopharmaceuticals, if proven successful, cost:benefit ratio will favor its medical use; Public perception and ethical considerations: stict regulations overseeing use of technology are required (danger than gene therapy could be used to 'improve' human characteristics, possibilty of eugenics is now almost a reality, safeguard to prevent this is already in place [restricted to somatic cells & genetic manipulation of human germ cells is banned])

expression of oncogenes, leading to transformed state, overexpression of cytokines associated worsening of disease symptoms, overproduction of angiotensinogen - ultimiately results in hypertension, intracellular transcription/translation of virally encoded genes, inappropriate gne (over) expression could be ameliorated/prevented if down-regulated, nucleotide-bsed approach to do this is 'antisense technology' (generation of short, ss stretches of nucleic acids, oligonucleotides are capable of binding to DNA [at specific gene sites] or to mRNA, binding prevvents expression of gene product by preventing transcription or translation)

watson-crick base complementary binding and higher-order secondary and tertiary structures of RNA (regions engaged in intramolecular are poor targets for antisense oligos, desirable to synthesize a nucleotide complementary to mRNA backbone)

"blind" or "shotgun" approach: synthesize large numbers of oligos targeted to various (often overlapping) regions, ability of each to block translation of mRNA is then directly assessed in vitro assay; Computer programs to predict its higher-order structure: approach remains to be optimized, translation initiation sites of mRNAs are often targets (free form secondary structure) (sequence homologies can exist within these sequences in unrelated genes, reduces specificity of blockin geffect, could lead to clinically significant side effects)

oligonucleotides may act as steric blockers (prevent proteins involved in translation from binding to sequences in mRNA); generation of duplexes likely allows targeting by intracellular RNases (enzyme is binds to RNA-DNA duplexes and degrades RNA portion, most synthetic antisense oligonucleotides are DNA based)

BCL-2 oncogene products (enhance cell survival via inhibition of apoptosis); survivin (generally not expressed in healthy tissue, expressed at high levels in many common cancer types, promotes ccell division and inhibits apoptosis); Clusterin (cytoprotective 'chaperone' protein, upregulation associated with various human cancers)

Likely specificity; relatively low toxicity; requirement for low levels of oligo inside cell; ability to manufacture oligos of speficied nucleotide using automated synthesizers

sensitivity to nucleases; very low serum half lives; poor rate of cellular uptake; orally inactive

do not appear to cross BBB, binding to serum proteins (provides repository, prevents rapid renal excretion)

Not fully understood; most are charged molecules; molecular mass up to 10-12 kDa; receptor-mediated endocytosis appears to be most common mehanism; passive diffusion may occur as well as possibly by endocytosis

3'-exonucleases, metabolic products are mainly excreted via urinary route, frequent IV infusions are required for product administration

Modification of ribose sugar found in repeat nucleotide structure: attachment of methyl or methoxy ethyl (increases product stability, increases product potency [by enhancing binding affinity for RNA], also decrease activation of RNaseH, may be overcome by chimeric phsphorothioate oligos [2'-modified sugar nucleotides are placed only at ends of molecule])

by direct chemical synthesis