GENE THERAPY

Arushe Tickoo

B. Tech Biotech

GENE THERAPY

• The term gene therapy describes any procedure intended to treat or alleviate disease by genetically modifying the cells of a patient. It encompasses many different strategies and the material transferred into patient cells may be genes, gene segments or oligonucleotides.

• The genetic material may be transferred directly into cells within a patient (in vivo gene therapy), or cells may be removed from the patient and the genetic material inserted into them in vitro, prior to transplanting the modified cells back into the patient (ex vivo gene therapy).

Gene therapy was first conceptualized in 1972.

The first approved gene therapy case in the United States took place on 14 September 1990, at the National Institute of Health, under the direction of Professor William French Anderson. It was performed on a four year old girl named Ashanti DeSilva. It was a treatment for a genetic defect that left her with ADA-SCID, a severe immune system deficiency. The effects were only temporary, but successful.

In January 2014, researchers at the University of Oxford reported that six people suffering from choroideremia had been treated with a genetically engineered adeno-associated virus with a copy of a gene REP1. Over a six month to two year period all had improved their sight. Choroideremia is an inherited genetic eye disease for which in the past there has been no treatment and patients eventually go blind

TYPES OF GENE THERAPY

1. Somatic Gene Therapy In somatic gene therapy, the therapeutic

genes are transferred into the somatic cells (non sex-cells), or body, of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations. Somatic gene therapy represents the mainstream line of current basic and clinical research, where the therapeutic DNA transgene (either integrated in the genome or as an external episome or plasmid) is used to treat a disease in an individual

Most of these trials focus on treating severe genetic disorders, including immunodeficiency, haemophilia, thalassemia, and cystic fibrosis. These disorders are good candidates for somatic cell therapy because they are caused by single gene defects. The replacement of multiple genes in somatic cells is not yet possible.

Germ cells will combine to form a zygote which will divide to produce all the other cells in an organism and therefore if a germ cell is genetically modified then all the cells in the organism will contain the modified gene. This would allow the therapy to be heritable and passed on to later generations

2. Germline gene therapy

In germline gene therapy, germ cells (sperm or eggs) are modified by the introduction of functional genes, which are integrated into their genomes.

CLASSES OF DISEASES

• Because the molecular basis of diseases can vary widely, some gene therapy strategies are particularly suited to certain types of disorder, and some to others. Major disease classes include:

• Infectious diseases (as a result of infection by a virus or bacterial pathogen);

• cancers (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

• Inherited disorders (genetic deficiency of an individual gene product or genetically determined inappropriate expression of a gene);

• Immune system disorders (includes allergies, inflammations and also autoimmune diseases, in which body cells are inappropriately destroyed by immune system cells).

CLASSICAL GENE THERAPY

• An essential component of classical gene therapy is that cloned genes have to be introduced and expressed in the cells of a patient in order to overcome the disease. Practically, this usually involves targeting the unaffected cells of diseased tissues.

• Immune system-mediated cell killing In many gene therapies the target cells are healthy immune system cells, and the idea is to enhance immune responses to cancer cells or infectious agents.

• Two major approaches for gene therapy: transfer of genes into patient cells outside of the body (ex vivo) or inside the body (in vivo).

IN-VIVO AND EX-VIVO GENE TRANSFER

In vivo gene therapy (blue arrow) entails the genetic modification of the cells of a patient in situ. Ex vivo gene therapy (black arrows) means that cells are modified outside the body before being implanted into the patient.

EX VIVO GENE TRANSFER

• Transfer of cloned genes into cells grown in culture.• Those cells which have been transformed successfully are

selected, expanded by cell culture in vitro, then introduced into the patient.

• To avoid immune system rejection of the introduced cells, autologous cells are normally used: the cells are collected initially from the patient to be treated and grown in culture before being reintroduced into the same individual.

• Clearly, this approach is only applicable to tissues that can be removed from the body, altered genetically and returned to the patient where they will engraft and survive for a long period of time (e.g. cells of the hematopoietic system and skin cells).

• This type of gene therapy involves transplantation of autologous genetically modified cells and so can be considered a modified form of cell therapy.

IN VIVO GENE TRANSFER

• The cloned genes are transferred directly into the tissues of the patient.

• This may be the only possible option in tissues where individual cells cannot be cultured in vitro in sufficient numbers (e.g. brain cells) and/or where cultured cells cannot be re-implanted efficiently in patients.

• Liposomes and certain viral vectors are increasingly being employed for this purpose.

• It is often convenient to implant vector-producing cells (VPCs), cultured cells which have been infected by the recombinant retrovirus in vitro: in this case the VPCs transfer the gene to surrounding disease cells.

• As there is no way of selecting and amplifying cells that have taken up and expressed the foreign gene, the success of this approach is crucially dependent on the general efficiency of gene transfer and expression.

MAMMALIAN VIRAL VECTORS BECAUSE OF THEIR HIGH EFFICIENCY

OF GENE TRANSFER

ONCORETROVIRAL VECTORS

• Retroviruses are RNA viruses which possess a reverse transcriptase function, enabling them to synthesize a complementary DNA form.

• Following infection (transduction), retroviruses deliver a nucleoprotein complex (pre integration complex) into the cytoplasm of infected cells.

• This complex reverse transcribes the viral RNA genome and then integrates the resulting DNA copy into a single site in the host cell chromosomes.

• Retroviruses are very efficient at transferring DNA into cells, and the integrated DNA can be stably propagated, offering the possibility of a permanent cure for a disease.

ONCORETROVIRAL VECTORS

• Because of these properties, retroviruses were considered the most promising vehicles for gene delivery and currently about 60% of all approved clinical protocols utilize retroviral vectors.

• Since all the viral genes are removed from the vector, the viruses cannot replicate by themselves.

• They can accept inserts of up to 8 kb of exogenous DNA and require a variety of packaging systems to enclose the viral genome within viral particles

• Oncoretroviruses can only transduce cells that divide shortly after infection: the pre integration complex is excluded from the nucleus and can only reach the host cell chromosomes when the nuclear membrane is fragmented during cell division.

gene therapy

ADENOVIRUS VECTORS

• Adenoviruses are DNA viruses that produce infections of the upper respiratory tract and have a natural tropism for respiratory epithelium, the cornea and the gastrointestinal tract.

• Adenovirus vectors have been the second most popular delivery system in gene therapy (with extensive applications in gene therapy for cystic fibrosis and certain types of cancer).

• They are human viruses which can be produced at very high titers in culture, and they are able to infect a large number of different human cell types including nondividing cells.

• Entry into cells occurs by receptor-mediated endocytosis and transduction efficiency is very high (often approaching 100% in vitro).

• They are large viruses and so have the potential for accepting large inserts (upto 35 kb).

ADENOVIRUSES ENTER CELLS BY RECEPTOR-MEDIATED

ENDOCYTOSIS

Binding of viral coat protein to a specific receptor on the plasma membrane of cells is followed by endocytosis. Subsequent vesicle disruption by adenovirus proteins allows virions to escape and migrate towards the nucleus where viral DNA enters through pores in the nuclear envelope.

DISADVANTAGES OF ADENOVIRUSES

• The inserted DNA does not integrate, and so expression of inserted genes can be sustained over short periods only.

• For example, the recombinant adenoviruses used in cystic fibrosis gene therapy trials showed that transgene expression declined after about 2 weeks and was negligible after only 4 weeks.

• Because they can infect all human cells, adenovirus vectors may be risky in some therapies that are designed to kill cancer cells without causing toxicity to normal surrounding cells.

• Most importantly, first generation adenovirus vectors can generate unwanted immune responses, causing chronic inflammation.

HERPES SIMPLEX VIRUS VECTORS

• HSV vectors are tropic for the central nervous system (CNS) and can establish lifelong latent infections in neurons.

• They have a comparatively large insert size capacity (>20 kb) but are non integrating and so long-term expression of transferred genes is not possible.

• Their major applications are expected to be in delivering genes into neurons for the treatment of neurological diseases, such as Parkinson's disease, and for treating CNS tumors.

LENTIVIRUSES

• The lentivirus family, which includes HIV (human immunodeficiency virus), are complex retroviruses that infect macrophages and lymphocytes.

• Unlike oncoretroviruses, lentiviruses are able to transduce non dividing cells.

• In the case of HIV, for example, the pre integration complex contains nuclear localization signals that permit its active transport through nuclear pores into the nucleus during interphase.

• Because of their ability to infect non dividing cells and to integrate into host cell chromosomes, considerable efforts are now being devoted to making lentivirus vectors for gene therapy.

MOST COMMON VIRAL VECTORS

Retroviruses

Adenoviruses

Adeno-associated viruses

Herpes simplex viruses

can create double-stranded DNA copies of their RNA genomes. Can integrate into genome. HIV, MoMuLV, v-src, Rous sarcoma virus

dsDNA viruses that cause respiratory, intestinal, and eye infections in humans. Virus for common cold

ssDNA viruses that can insert their genetic material at a specific site on chromosome 19

dsDNA viruses that infect a neurons. Cold sores virus

METHODS OF GENE DELIVERY (THERAPEUTIC CONSTRUCTS)

-- Injection of DNA via Mechanical and electrical strategies include microinjection

-- DNA transfer by liposomes (delivered by the intravascular, intratracheal,

intraperitoneal or intracolonic routes)

-- DNA coated on the surface of gold pellets which are air-propelled into the epidermis

(gene-gun), mainly non applicable to cancer

-- Biological vehicles (vectors) such as viruses and bacteria. Viruses are genetically engineered

so as not to replicate once inside the host. They are currently the most efficient means of gene transfer.

MECHANICAL AND ELECTRICAL TECHNIQUES

• Mechanical and electrical strategies of introducing DNA into cells include microinjection, particle bombardment, the use of pressure, and electroporation.

• The direct injection (microinjection) of naked DNA (i.e., uncomplexed DNA) into a cell nucleus is perhaps the most simple, and therefore appealing, approach to gene delivery.

BIOLISTIC DNA INJECTION (GENE GUNS)

Invented for DNA transfer to plant cells

Fully applicable to eukaryotic cells

plasmid DNA shown here

BIOLISTIC PARTICLE DELIVERY

• Particle bombardment, which is also called biolistic particle delivery, can introduce DNA into many cells (including cell-walled plant cells) simultaneously.

• In this procedure, DNA-coated microparticles (composed of metals such as gold or tungsten) are accelerated to high velocity to penetrate cell membranes or cell walls.

• Bombardment is widely employed in DNA vaccination, where limited local expression of delivered DNA (in cells of the epidermis or muscle) is adequate to achieve immune responses.

• Because of the difficulty in controlling the DNA entry pathway, this procedure is applied mainly adherent cell cultures and has yet to be widely used systemically.

ELECTROPORATION

• An alternative approach is to use high-voltage electrical pulses to transiently permeabilize cell membranes, thus permitting cellular uptake of macromolecules.

• This process, called electroporation, was first used to deliver DNA to mammalian cells in 1982 .

• Since that time, electroporation has been used to deliver DNA to myriad cell types in vitro, including bacteria and yeast.

• It is one of the most efficient gene transfer methods, but it is limited because of the high mortality of cells after high-voltage exposure and difficulties in optimization.

• Although electroporation is difficult to apply in vivo, some progress has been achieved in skin, corneal endothelium, and muscle.

CHEMICAL METHODS

• The use of uptake-enhancing chemicals-which is arguably the easiest, most versatile,most effective, and most desirable of the DNA delivery methods—was demonstrated more than 30 years ago.

• The general principle is based on complex formation between positively charged chemicals (usually polymers) and negatively charged DNA molecules.

• These techniques can be broadly classified by the chemical involved : 2-(diethylamino) ether (DEAE)-dextran, calcium phosphate, artificial lipids, protein, dendrimers, or others.

DEAE DEXTRANAND CALCIUM PHOSPHATE

• DEAE dextran and calcium phosphate, which interact with DNA to form DEAE-dextran–DNA and calcium phosphate–DNA complexes, respectively.

• After the complexes are deposited onto cells, they are internalized by endocytosis.

• DEAE-dextran and calcium phosphate methods are simple, effective, and still widely used in the laboratory for in vitro transfection.

• Even so, both methods are hampered by cytotoxicity and the difficulty of applying them to in vivo studies. In addition, DEAE dextran can be used neither with serum in culture medium nor for stable transfection.

• The calcium phosphate method also suffers from variations in calcium phosphate–DNA sizes, which causes variation among experiments.

LIPOSOMES

Lets’ wrap it in something safe to increase transfection rate

Therapeutic drugs

Lipids – are an obvious idea !

DNA DELIVERY OF GENES BY LIPOSOMES

Cheaper than viruses

No immune response

Especially good for in-lung delivery (cystic fibrosis)

100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector

Complexation of cationic lipids with DNAwas first described in 1987, and ‘lipofection’was reported to be 5- to >100-fold more efficientthan the earlier calcium phosphate or the DEAEdextran transfection techniques. Cationic lipids are highly soluble in aqueous solution, forming positively charged micellar structures termed liposomes

LIPOFECTINS

• Felgner and colleagues developed the cationic lipid Lipofectin in 1987.

• Lipofectin–DNA complexes can be handled easily and, therefore, became one of the first chemical systems that could be used in animals.

• In addition, DNA has been successfully complexed with cationic, anionic, and neutral liposomes, as well as various mixtures.

LIPOSOMES

• Lipid based systems are probably the most commonly used methods of DNA delivery and have been used in human clinical trials.

• Still, lipid based systems have important drawbacks, including the lack of targeting, the poorly understood structure of DNA–lipid complexes, and variations arising during fabrication.

• The major limitation of the above approaches is toxicity upon systemic administration.

• Furthermore, a major problem with the application of most nonviral systems, including lipoplexes, is their poor efficiency at transfecting nonproliferating cells. This is thought to be mainly a result of the integrity of the nuclear membrane providing a physical barrier to entry.

• Lipoplexes are seen with diameters of 100–200 nm, and also elongated, ‘spaghetti’-shaped, lipoplexes. Large aggregates or ‘meatball’ lipoplexes are also observed, and thought to comprise numerous lipid and DNA molecules.

POLYETHYLENIMINE

• PEI is a branched polymer with high cationic potential that is capable of effective gene transfer in nondifferentiated COS-1 cells; however, it can also be extremely cytotoxic due to induction of apoptosis.

• The high transfection efficiency of PEI can be attributed to the buffering effect or the “proton sponge effect” of the polymer caused by the presence of amino groups in the molecule.

• The strong buffering effect of the polymer helps in rapid endosome escape.

• The cytotoxicity and transfection efficiency of PEI are directly proportional to its molecular weight.

• Efforts to reduce the toxicity by synthesis of PEI with graft copolymers such as linear poly(ethylene glycol), incorporation of low molecular weight PEI, and PEI glycosylation are under way.

Fig; Gene Therapy of mouse showing modification in its tail by removing the defected gene or replacing the defected gene with the functional gene

APPLICATION