ࡱ> 7 2+bjbjUU 5m7|7|&$lvvvvtp"p"p"8# D#l'D#####'''CCCCCCC$E HTC'''''C*vv##C***'4v##C*'C**1A"lC## ;xnp"*BCC0'DBcH*cHC*vvvvNon Viral Gene Therapy Aims of Lecture: Introduction & brief overview of Gene therapy Aims of gene therapy and barriers to success Introduce the main non-viral delivery systems Goal for Gene therapy: To permanently cure inherited diseases by the replacement of a defective gene with a fully functioning one. The Need for Gene Therapy Thousands of single gene inherited disorders are known. Few can be treated effectively - usually only symptomatic relief is possible Have a large effect on quality of life of sufferers e.g. study of 351 major Single gene Disorders. Current treatment of each disorder assessed by 3 different outcomes: Assessment Normal lifespan Reproduction possible Social adaptation satisfactory % Success 15 11 6 Genetic disorders are therefore extremely debilitating ( Taken from the report of the Committee on Ethics of Gene Therapy 1992, UK) Gene therapy could also be used to treat some chronic diseases e.g viral infections, cancer diabetes gene vaccination for immunisation All involve delivery, to target cells, of an expression cassette made up of one or more genes and the sequences controlling their expression. Reminder: Gene therapy and genetic engineering raises considerable feeling amongst the population. There are ethical issues involved in gene therapy which can affect choice of methodology and its uses. DNA DELIVERY Molecular Biology is now increasingly understood, but the main problem is of delivering the DNA to the relevant cells in such a way that the gene product is expressed at a therapeutic level. At present this would involve inserting a normal gene while leaving abnormal gene in place Ideally want gene integrated into normal place in genome and transmitted to cells progeny each time cell divides. In practice: DNA is inserted at a random site in DNA. Most genes function satisfactorily in this way - but may lead to: Inappropriate control of the gene May disrupt some other genetic function - with unwanted consequences Alternatively plasmid or DNA may exist in nucleus separate from chromosome i.e. episomal - wont disrupt genetic function, but relatively easily lost from nucleus - will not necessarily be replicated and transferred equally to daughter cells Strategies For Gene Therapy A number of different strategies are possible for gene therapy. These strategies are general and could be used with any method of transferring DNA to cells. A. In vitro Gene transfer protocols 1. Remove host cells 2. Expand by cell culture 3. Transfect with new gene 4. Establish stable transfection 5. Return to host Main indications: Defects affecting blood cells, e.g. severe combined immune deficiency due to adenosine deaminase malfunction Some possibilities using fibroblasts, hepatocytes and myoblasts, but involve greater difficulties than with blood cells but: Difficult to get prolonged gene expression and establish a genetically altered cell population Number of suitable target diseases is limited. B. In vivo gene transfer protocols Use of any of the normal parenteral delivery routes for gene therapy either using direct transfer, viral or non-viral vectors. Has all the problems of in vitro delivery, and in addition has to fulfil following criteria: Avoid uptake of RES Avoid immunogenic responses if more than one administration is required. Target to relevant organ or tissue may be possible to overcome this by using tissue specific promoter sequences. Barriers to DNA delivery to the cell: Barrier 1. Cell membrane 2. Escape from endosome 3. Translocation across cytoplasm 4. Entry into nucleus 5. Prevention of gene loss 6. Expression of gene product Comments Condensation of DNA. Small size is important for endocytosis Pore formation/endosomal disruption required Diffusion is very slow. Active mechanisms involving cytoskeleton may be used Transport system for nucleic acids through nuclear pores in nuclear membrane. Ideally gene should be inserted into chromosome but episomal expression used Vectors DNA is a large, long, fragile molecule easily degraded by serum nucleases and intracellular nucleases. Therefore needs to be compacted and protected. Delivery is aided by a vector which delivers cassette to intracellular site. Viral Vectors Where some of the DNA involved in viral replication is removed and replaced by a gene cassette. Viral vectors are very efficient at transfecting cells have evolved machinery to deliver DNA effectively. However there are worries about: Disabled viruses regaining virility, Viruses are very immunogenic Viral therapy has resulted directly in a death and two cases of leukemia. Non-Viral Vectors Non-Viral vectors easier to manufacture, scale-up and quality control Becomes like any other drug delivery problem - familiar ground for the pharmaceutical industry. Aim of non-viral delivery To deliver a piece of DNA efficiently to the nucleus of appropriate cells where it will become an extrachromosomal element (episome) capable of generating gene products for a discrete period of time before elimination from the host. i) Physical e.g. microinjection, electroporation ii) Delivery systems e.g. cationic lipids, cationic polymers PHYSICAL METHODS OF GENE INSERTION Direct injection: Injection directly into skeletal of cardiac muscle can be effective in expression of some recombinant genes. formation of myotubes and transfer of DNA - no evidence of DNA integration but expression persisted for 6 months Microparticle bombardment (gene gun): DNA adsorbed onto submicron sized gold or tungsten particles. Particles accelerated to high velocity using a gas discharge - Expression observed following bombardment of skin, muscles, liver, intestine and mammary gland - may be particularly useful for gene vaccination similar technique can be used on cells Electroporation: Cells exposed to very high electric field strengths in the presence of gene or plasmid Blasts temporary holes in cell membrane Allows passage of DNA into the cytoplasm - Has been used in vivo as well These physical methods are likely to be particularly useful for DNA vaccination, but their role in other forms of gene therapy is unclear NON-VIRAL GENE DELIVERY THROUGH PARTICULATE SYSTEMS General Aim: To produce easy to assemble particles with virus-like activity.  2 Main Areas of research both based on charge neutralisation of DNA: Cationic lipids Cationic polymers Cationic Lipids: Lipids have been synthesised with a cationic headgroup which can interact with the negatively charged DNA. A range of novel lipids have been designed e.g. DOTMA (dioleoyloxypropyl-trimethylammonium bromide) . These are mixed with neutral lipids such as DOPE and cholesterol, and the DNA to give a lipid-DNA complex. Sometimes referred to as Cationic liposomes, but the DNA binds to the surface of the lipid rather than being encapsulated.- Can be obtained commercially as a ready to use mixture of lipids, e.g. lipofectin - up to 90% transfection of cells in vitro has been obtained - has been used routinely in vivo as well as in vitro, e.g. for therapy of alpha-1 antitrypsin and cystic fibrosis conductance regulator (CFTR) deficiency. Cationic Polymers ( complexes) Based on polyelectrolyte theory that oppositely charged polymers assemble spontaneously into small particles. Cationic polymers condense DNA into small particles in range 30-200nm. - cationic polymers protect DNA from degradation Much initial work done on Poly l-lysine because - Cationic polymers enhance DNA uptake - Poly-L-Lysine (PLL) enhances uptake of many substances due to +ve charge leading to adsorption on negative cell surfaces (adsorption) - promotes endocytosis Now many other types of polymers used e.g. chitosan, amino methacrylates, polyamidoamines, other cationic polyamino acids, polyethylenimine, various cationic dendrimers. Additional functionality now included in complexes to address delivery problems: a. Uptake of complexes Targeting ligands e.g. peptide sequences, folic acid, sugar moieties, endocytosed proteins b. Incorporation of polyethylene glycol (PEG) on particle surfaces to avoid opsonisation for in vivo use c. Endosomolytic functions i. use of polymers designed to destabilise endosomes by proton sponge mechanisms ii. incorporation of endosomolytic peptides which create pores in membranes. d. Incorporation of nuclear localisation peptide sequences to facilitate passage across the nuclear membrane. Example of some early work on incorporating some of these principles: Uptake of PLL-DNA complexes enhanced by targeting ligands, e.g. asialoglycoprotein (ASGP) or transferrin linked to PLL DNA expression still poor - only about 1/5th that of lipofectin. Addition of fusogenic proteins - Fusogenic proteins are negatively charged and therefore incorporated into PLL-DNA complexes - Disrupt endosomal membranes and allow transfer of DNA from endosomal compartment to cytoplasm - promote massively increased gene expression of PLL-DNA complexes These components give a general design for an efficient non-viral DNA delivery system: Cationic polymer condenses DNA, and protects it from degradation Ligands give tissue specificity and enhance uptake Fusogenic peptides allow escape from endosomal compartment  SUCCESS OF GENE THERAPY APPLICATIONS Gene therapy is very complex in terms of: Understanding how genes are controlled and function efficiently in producing their gene products Understanding intracellular processes involving translocation of macromolecules like DNA Understanding complex colloidal chemistry in how to build effective stable particles Understanding how to incorporate appropriate biological signals to make the complex function effectively like a virus. - gene therapy is not yet well understood. - more basic science on vectors required before gene therapy will reach its objectives MCG 2003 MCG 2003 *;U[ i vUgjYiY z `!a!!g&&&((((((***++ +2+CJOJQJ j)U>* PJmH sH jPJUmH sH \5CJ 56\]55\5CJ @*+Y:;U ]^ & F$a$* +1+&'tuY Z [ i j ) * $a$`^*  t u 8  ]^` ]^]  & F] ]^ 23E01de D]  & F ]  & F ]  & F] ]^"#$:]:;VWuv;<|8 ]^]8 STUgh)G*V ]^]  & F] ]^Ahjkl6 % &1$ ]^` ]^ ]^]6FXYj=>?@{Y Z [ z /!a!!!C"["^"# #Z# ]^ ]^]Z#[#r###7$8$S$$$$d%e%%%#&$&e&f&g&&&&H'I'''] ]^ ]^''''(Z(((((((((((M)))r*s***++ + $]^a$  & F] ]^] + + +++++++++++++++++++ +!+"+#+$+%+&+'+(+(+)+*+++,+-+.+/+0+1+2+ ]^ #0P|. 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