Therapeutic Nucleic Acids

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gen terapisi, gene therapy, bioteknology, biyoteknoloji, vectors, gen tedavisi, gen transferi, ex vivo, in vivo

Transcript of Therapeutic Nucleic Acids

Therapeutic Nucleic Acids

Chapter 2Therapeutic Nucleic Acids 1This chapter describes in an analytical manner the various types of molecules that are part of gene therapy. These can be grouped into one of two classes: DNA sequences coding for proteins having various cellular functions; nucleic acids (DNAs or RNAs) with regulatory function, either synthesized as small synthetic molecules or, in the case of RNAs, expressed inside the cells after transfer of the corresponding genes.

2.1 Protein-Coding GenesThe concept of gene therapy was originally developed with the idea of supplying a missing cellular function by transferring a normal copy of the altered gene into the relevant cells. 2.1 Protein-Coding GenesIn reality, in human cells the average size of protein-coding genes is 27 kb, by far longer than the maximum length fitting the most common gene delivery systems. For this reason, gene therapy is most commonly based on the transfer of cDNAs average length: 2.5 kb or of their protein-coding portion (average length: 1.5 kb, corresponding to about 500 codons. 2.1 Protein-Coding GenesFrom the molecular point of view, the transfer of a gene, or its cDNA or its cDNA coding region has essentially different properties. Both cDNAs and their coding portions need to be transcribed from promoters that are usually different from the natural ones, which are commonly too large to be used. 2.1 Protein-Coding GenesIn addition, the cDNA coding portions alone lack the regulatory elements controlling gene expression at the post-transcriptional level, which are usually contained in the introns or in the untranslated regions (UTRs) at the 3' and 5' ends of the cDNAs. These regions are commonly involved in the regulation of cDNA stability, transport, subcellular location and translation of the cellular mRNAs.Protein-Coding Genesin several instances the levels at which proteins are produced are not very important, and thus a tight translational or posttranslational regulation of gene expression is not required. For example, this is the case of proteins replacing missing cellular functions in the hereditary disorders of metabolism, or antigens for anti-cancer vaccination, or secreted antibodies. In these cases, transfer of the protein-coding region under the control of a strong promoter, such as the promoter for the cytomegalovirus (CMV) immediate-early (TE) genes, is adequate. Protein-Coding GenesIn several circumstances, inclusion of a small intron upstream of the cDNA coding sequence, or of a 3' UTR downstream of it, are known to facilitate expression of the protein of interest. The proteins encoded by the therapeutic genes can have very different functions, ranging from the substitution of a missing cellular protein to the modulation of the immune system.

2.1.1 Proteins Substituting Missing or Mutated Cellular Proteinsgene therapy was conceived with the purpose to express proteins that are missing or defective, thus curing autosomal recessive and X linked disorders. These replacement proteins can exert their functions inside the cells (for example, in the case of gene therapy for muscular dystrophies) or on the cell membrane (for example, the CFTR gene in cystic fibrosis), or be secreted into the extracellular environment or the blood stream (as is the case of coagulation factors in the hemophilias).

2.1.2 Proteins Modulating Cellular Functions The objective of several gene therapy applications is not to supply a missing cellular function, but to express proteins able to modulate cell behavior. The approaches utilized are very variegate. 2.1.2 Proteins Modulating Cellular Functions For example, in the cancer gene therapy field several experimentations take advantage of the possibility to induce arrest of cell proliferation using CDK inhibitors such as p27 or p21, or checkpoint proteins such as p53. Again in the cancer gene therapy field, another interesting class of proteins is those that increase the therapeutic index of chemotherapy.

2.1.2 Proteins Modulating Cellular Functions On several occasions, myeloid toxicity limits the dose of antineoplastic drugs that can be administered to a patient with a solid cancer. In these cases, it is possible to confer resistance to CD34+ hematopoietic precursors by transferring into these cells a gene coding for a membrane transporter, such as the mdr gene, able to prevent the intracellular accumulation of a large series of anticancer drugs.

2.1.2 Proteins Modulating Cellular Functions Other examples of proteins modulating cellular functions are those used for gene therapy of viral infections, which block activity of some viral proteins and thus impair viral infection. Among these proteins, RevM10 is a mutated form of the HIV-l Rev protein, acting as a trans dominant negative mutant: when this protein is present in CD4+ T lymphocytes, it blocks wild-type Rev function and thus inhibits viral infection.

the immune response against cancer cells can be increased by expressing, into these cells, the genes coding for co-stimulatory proteins, such as B7, ICAM-1, or LFA-3, which are commonly downregulated in tumors as a mechanism of immune escape and are however necessary for proper antigenic presentation to cytotoxic T lymphocytes (CTLs). Cytotoxic T lymphocytes Lymphocytes that kill other ("target") cells.Targets may include:virus-infected cellscells infected with intracellular bacterial or protozoal parasitesallografts such as transplanted kidney, heart, lungs, etccancer cells2.1.3 Secreted Growth Factors and CytokinesA number of gene therapy applications are based on the delivery of genes coding for secreted factors and cytokines, having various activities. For example, in cancer gene therapy, several clinical approaches take advantage of the genes coding for interleukin-2 (IL-2), lL- 12, IL-7, IL-4, GM-CSF, and other cytokines to increase antigen stimulation or modulate the immune response against the transformed cells. 2.1.3 Secreted Growth Factors and CytokinesIn the field of cardiovascular disorders, vascular endothelial growth factor VGF gene transfer is used by a number of applications aimed at the induction of therapeutic anginogenesis in patient with cardiac or peripheral iskemia, due to the powerful activity of this factor in inducing new blood vessel formation.

2.1.3 Secreted Growth Factors and CytokinesFinally, gene therapy for a number of neurodegenerative disorders is based on the delivery of genes coding for neurotrophic factors, including nerve growth factor (NGF) in Alzheimer's disease, neurturin (NTN) in Parkinson's disease and ciliary neurotrophic factor (CNTF) in Huntington's disease.

Huntington'sbrainnormalbrain2.1.4 Proteins Regulating Cell Survival and Apoptosis Several pathologic conditions are caused by the death of some cell types or, on the contrary, by inappropriate survival of others. 2.1.4 Proteins Regulating Cell Survival and Apoptosis Examples of the former condition are neurodegenerative disorders, which are caused by accelerated apoptotic death of some neuronal populationsin contrast, tumors represent a paradigmatic example of a condition in which lack of cell apoptosis essentially contributes to disease development.

2.1.4 Proteins Regulating Cell Survival and Apoptosis Since apoptosis ensues as the net result of the function of various proteins favoring or contrasting this processtransfer of the genes coding for these proteins might have a therapeutic role. 2.1.4 Proteins Regulating Cell Survival and Apoptosis For example, one of the gene therapy approaches for amyotrophic lateral sclerosis (ALS), a motoneuron disorder, is based on the transfer of the hc/-1 gene, coding for an anti-apoptotic protein.

2.1.4 Proteins Regulating Cell Survival and Apoptosis In contrast, apoptosis in tumors can be induced by oligonucleotides targeting a few antiapoptotic proteins, including Bcl-1 itself, Survivin, or the X-linked inhibitor of apoptosis (XIAP).

2.1.4 Proteins Regulating Cell Survival and Apoptosis Another way to interfere with cell survival is the so-called "suicide gene" approach. Cells are transduced with a vector expressing the thymidine kinase gene of the herpes simplex virus type 1 (HSV-TK), an enzyme that is innocuous per se but becomes toxic in the presence of the drug gancyclovir. The result of this activation is a block in DNA synthesis and consequent cell death by apoptosis.

2.1.5 Antigens for Vaccination

2.1.5 Antigens for VaccinationA vast range of gene therapy applications have the immune system as their target.These consist in the utilization of gene transfere to activate the immune system, by either the delivery of genes coding for immunomodulatory cytokines ortransferring, into cancer cells, genes coding for the co-immunostimulatory proteins necessary for antigenic presentation. 2.1.5 Antigens for VaccinationA growing field of gene therapy applications in this are is that of DNA-based vaccination (genetic vaccination). This is based on the in vivo delivery of genes usually coding for viral or tumor cell antigens, to seek activation of the immune system against the encoded proteins. 2.1.5 Antigens for VaccinationThe antigen gene can be transferred using naked plasmid DNA "DNA vaccination" sometimes using physical methods such as gene transfer facilitators (for example, the "gene gun" approach), or viral (adenovirus, vacciniavirus)2.1.5 Antigens for VaccinationGenetic vaccination offers several advantages over the common vaccination strategies based on the administration of either attenuated or inactivated microorganisms, or on protein antigens. 2.1.5 Antigens for VaccinationThese include the possibility to evoke a cytotoxic immune response, since the processed exogenous proteins are directly expressed in the context of MHC Class I, together with the relative safely, reduced cost, and prolonged storage. Cancer and HIV-1 infection are among the disorders in which genetic vaccination is most commonly considered.

2.1.6 Antibodies and Intracellular AntibodiesA peculiar class of therapeutic genes are those coding for antibodies. Natural antibodies have a typical Y-shaped structure, composed of 4 polypeptide chains: two identical heavy (H) chains ( -440 amino acids each) and two identical light (L) chains (- 220 amino acids).Both the H and L chains contain a variable region V (V H and V L), which together recognize the antigen.

antigen2.1.6 Antibodies and Intracellular AntibodiesThe H chains then contain three constant (C) regions (CHI, CH2, and CH3) and a flexible hinge (h) region between CH 1 and CH2, while the L chains only have one constant region (CL)The H and L chains are synthesized separately in the B-cell endoplasmic reticulum and assemble thanks to the formatton of both inter- and intra-chain disulfide bonds.2.1.6 Antibodies and Intracellular Antibodies Digestion of an antibody molecule with the proteolytic enzyme papain generates three fragments, which can be separated by chromatography.

2.1.6 Antibodies and Intracellular AntibodiesTwo of these are identical and correspond to the antigen-binding portion of the antibody (antigen-binding fragment, Fab), composed of the whole L chain (VL +CL) and the N-terminal portion of the H chain, including the variable region (VH) and the CH1 constant region.

2.1.6 Antibodies and Intracellular AntibodiesThe third fragment (crystallizable fragment, Fc) is instead only composed of the C-terminal region of the H chain, which is able to bind complement and different receptors, AnFc receptoris a protein found on the surface of certain cells - includingB lymphocytes,natural killer cells,macrophages,neutrophils, mast cells- that contribute to the protective functions of theimmune system.

2.1.6 Antibodies and Intracellular AntibodiesThe fact that an antibody consists of four polypeptide chains poses obvious problems for its expression by gene transfer. However, it is possible to obtain synthetic antibodies consisting of a single polypeptide chain formed by the V portions of the H and L chains (Vh and VL) of the natural antibodies, separated by the flexible amino acid linker2.1.6 Antibodies and Intracellular AntibodiesThis construct, named single-chain Fv (scFv), contains all the structural determinants allowing specific binding of the molecule to its target antigen. While the mass of a natural IgG antibodys about 150 kDa, that of a scFv is 29 kD, corresponding to about 250 amino acids.

2.1.6 Antibodies and Intracellular AntibodiesThe Vh and VL regions contain three hypervariable regions that essentially participate in antigen recognition (CDR).An even simpler form of antibody only contains the CDR regions of one chain. 2.1.6 Antibodies and Intracellular AntibodiesThese are named single domain antibodies, with analogy to the antibodies produced by some camelids, in which the antigen-binding site is composed of a single chain, analogous to the mammalian H chain.

2.1.6 Antibodies and Intracellular AntibodiesNeither scFv nor single-domain antibodies exert any effector activity (compment fixation, binding to cellular receptors), since these are commonly associated with the Fc portion of natural antibodies. The simplest manner to obtain an scFv antibody is to clone the Vh and VL region of a monoclonal antibody (mAb) having high affinity for the antigen of interest. 2.1.6 Antibodies and Intracellular AntibodiesAlternatively, the scFv can be obtained by the direct screening of an scFv library, for example a phage display library, in which the antibody is displayed on the phage surface. The scFv-coding gene can thus be cloned into a plasmid or a viral vector and transferred into the cell.

2.1.6 Antibodies and Intracellular AntibodiesWhen the scFv gene coding sequence is preceded by a secretion signal, the antibody is secreted outside the cells. In order to stabilize an scFv antibody and increase its avidity the VL-VH region can be followed by a portion of the antibody Fc fragment. This can induce dimerization of two scFvs and, formation of disulfide bonds. These types of engineered antibodies are known as minibodies.

2.1.6 Antibodies and Intracellular AntibodiesNatural antibodies are secreted in serum or expressed on the surface of B lymphocytes and recognize their target antigens only when these are present in the extracellular environment or expressed on the cell surface. In the absence of a secretion signal, the scFv antibodies can instead be translated in the cytosol. 2.1.6 Antibodies and Intracellular AntibodiesIn this reducing compartment, disulfide bonds cannot be formed, however some scFvs can still fold properly in the absence of these bonds and retain their binding specificity. These intracellular antibodies, also named intrabodies, thus have the important potential to target intracellular antigens.

2.1.6 Antibodies and Intracellular AntibodiesBoth scFv and single-domain intracellular antibodies can be used for a vast series of therapeutic applications. They can block interaction between two intracellular proteins, or protein binding to DNA, or inhibit function of an enzyme. Alternatively, they can re-localize a protein to a cellular compartment different from its normal, thus blocking its activity. For example, intracellular antibodies having a nuclear localization signal can bind a cytosolic antigen and relocalize it into the nucleus.2.1.6 Antibodies and Intracellular AntibodiesA vast series of pre-clinical studies have shown the potential efficacy of intracellular antibodies for gene therapy of various disorders. In the field of gene therapy for viral infections, the cell can be rendered resistant to infection by intracellular antibodies blocking function of the proteins, of either viral or cellular origin, that are essential for viral replication. 612.1.6 Antibodies and Intracellular AntibodiesIn gene therapy of cancer, intracellular antibodies are used to modify the intracellular localization, and thus inhibit the function, of oncogenes such as ErbB-2 in breast and ovary carcinoma.

2.1.6 Antibodies and Intracellular AntibodiesIn the field of gene therapy for neurodegenerative disorders, the use or intracellular antibodies can block the accumulation of pathological proteins, such as Tau in Alzheimers disease or the PrPsc protein in prion disease.

2.1.7 T-Cell Receptor (TCR) SubunitsAn interesting class of protein-coding genes is that including variants of the T-cell receptor (TCR), used to modify the target specificity of T-lymphocytes. In both viral infection and cancer a potentially powerful therapeutic approach would consist in the ex vivo activation of the patients CD8+ CTLs reacting against specific viral or tumor antigens, followed by their reinfusion in vivo, a procedure known as adoptive immunotherapy.

2.1.7 T-Cell Receptor (TCR) SubunitsMore efficacious than recovering the endogenous antigen-specific CTLs, which are usually limited in number, this can be achieved by modifying the specificity of any CTL by transducing these cells with the genes coding for a TCR of choice. The TCR consists of a complex of at least 6 different proteins.Antigen recognition specificity is conferred by a heterodimer of a/b (in most cases) or g/d chains.

T-Cell Receptor (TCR) Subunits2.1.7 T-Cell Receptor (TCR) SubunitsThe simplest manner to confer TCR specificity to T lymphocytes is to transfer, into these cells, the genes coding for the a and b chains specific for an antigen of interest obtained from a previously selected natural cell clone. Gene transfer is commonly achieved using a retroviral vector in which the genes coding for the two chains are separated by an IRES. A potential limitation of this approach, however, is that the exogenously introduced TCR molecules might complex with those endogenously expressed by the T cells, thus generating TCRs with different, and potentially undesired, target specificity. 2.1.7 T-Cell Receptor (TCR) SubunitsAn effective manner to overcome these problems is to exploit alternative ways to modify TCR specificity. One of these consists in the use of single-chain TCRs, in which mispairing between a and b chains is prevented. Another possibility is offered by the so called T-bodies. 2.1.7 T-Cell Receptor (TCR) SubunitsThese consist of a genetic fusion between a single-chain antibody recognizing the antigen of interest and the TCR CD3 zeta chain, able to activate the signal transduction cascade leading to cell activation. This approach both renders the engineered CTL independent from MHC restriction and prevents mispairing between the exogenous and endogenous TCR a and b chains.

http://nptel.ac.in/courses/102103041/21http://www.intechopen.com/books/gene-therapy-applications