Gene therapy

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Gene therapy is a method for the treatment or prevention of disease that uses genes to provide the patient’s somatic cells with the genetic information necessary to produce specific therapeutic proteins needed to correct or to modulate a disease.


Gene therapy

Gene therapy is a method for the treatment or prevention of disease that uses genes to provide the patient’s somatic cells with the genetic information necessary to produce specific therapeutic proteins needed to correct or to modulate a disease. The promise of gene therapy is to overcome limitations associated with the administration of therapeutic proteins, including low bio-availability, inadequate pharmacokinetic profiles, and high manufacturing cost. Gene therapy approaches are utilized for treating genetic and acquired diseases.

Two approaches are currently used for gene transfer: viral and nonviral. Viral gene transfer utilizes a genetically modified natural virus with a part of the viral genome replaced by a therapeutic gene (called transgene) and making the virus replication deficient. Viruses have evolved to efficiently penetrate cells and transfer their genetic material into host cells (a process called transduction). Thus, a viral vector efficiently transfers the desired genetic material into target cells, leading to transgene expression. Several different viral vectors have been developed for gene therapy, including ret-rovirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus (HSV). The advantages and disadvantages of different viral vectors are listed in Table 26.1.

Table 26.1 Characteristics of viral vectors


AAVs are the most common viral vectors used in the clinic. This is mainly due to their low safety risk compared to other viral vectors. Adenoviruses and AAVs do not integrate their genes into the host cell genome. Retroviruses, on the other hand, integrate their genetic material with the host cell genome. Thus, the duration of gene expression is much longer with retroviruses (weeks to months) as compared to adenoviruses and AAVs (days to weeks).


Retroviral vector

Retroviral vectors are RNA viruses (i.e., their genome is RNA) possessing the main feature of reverse transcribing their viral RNA genome into a double-stranded viral DNA. Retroviral vectors can stably insert into the host DNA. The retroviral genome consists of three encoding regions (por-tions of DNA strand that code for specific functional proteins) responsible for viral replication: (1) gag region, encoding group-specific antigens and proteins; (2) pol region, encoding reverse transcriptase; and (3) env region, encoding viral envelope protein. These regions are flanked on either side by a long terminal repeat (LTR) region. LTRs are responsible for regulation and expression of the viral genome.

These vectors can carry foreign genes of <8 kb (kilo base pair length). They carry an inherent risk of mutagenesis by inserting their genome (called insertion mutagenesis) within a functional gene, which can compromise the functionality of a critical normal human protein.

Defective retroviral vectors are devoid of the genes encoding viral pro-teins but retain the ability to infect cells and insert their genes into the chromosomes of the target cells. Members of this class include the Moloney murine leukemia viruses (MuLVs) and the lentiviruses.

1. MuLV

MuLV consists of three functional genes: gag, pol, and env, flanked by the viral LTRs. Removing these structural genes and inserting therapeutic genes in their place make muLV-based vectors. MuLV-derived vectors inte-grate exclusively in dividing cells.

2. Lentiviruses

The human immunodeficiency virus (HIV) is a lentivirus and is known to cause AIDS. Their special ability to infect and integrate into nondividing cells has application for the construction of lentiviral vectors for gene deliv-ery into nondividing, terminally differentiated cells such as neuronal tissue, hematopoietic cells, and myofibers.


Adenoviral vectors

Adenoviruses are nonenveloped DNA viruses carrying linear double-stranded DNA of about 35 kb length. The base pair length genome carrying capacity of a virus is limited by the size of genome that can be accommodated within the capsid. Adenoviral vectors infect both dividing and nondividing cells. Adenoviral vectors do not integrate into the host cell chromosomes. Genes introduced into cells using adenoviral vectors are maintained extrachromosomally in the nucleus and provides transient transgene expression.

For producing transgene-containing therapeutic adenoviruses, their genome is modified by deletion of the viral replication specific gene known as early gene 1 (E1A). This also creates space for the insertion of the desired gene. Adenoviral vectors are based on natural adenoviruses of serotypes 2 and 5. In these first generation adenoviral-vectors, additional partial dele-tions of E1B and E3 genes can be made to create more space for transgene insertion.

An advantage of adenoviruses over retroviral vectors is achievement of very high viral titers. This suggests efficient gene transfer. Their key disad-vantages are their episomal (extrachromosomal) status in the host cell that permits only transient expression of the therapeutic gene. Furthermore, expression of the E2 viral protein provokes inflammatory reactions and toxicities that limit repeated application of adenoviral vector for therapeu-tic benefit.


Adeno-associated virus vectors

AAV is a single-stranded DNA virus and belongs to the family of parvovi-ruses. For example, AAV-2 is a nonpathogenic human virus, and the wild type AAV-2 genome establishes a latent infection in human cells, where the viral genome integrates into the chromosomal DNA in a site-specific manner. AAV requires an adenovirus or a herpes virus for viral replica-tion. Compared to adenoviruses, AAV has low immunogenicity. It has a limited capacity for insertion of foreign genes ranging only from 4.1 to 4.9 kb. For construction of rAAV-based vectors, the rep and cap genes (which are responsible for the production of proteins that would replicate the virus or produce structural proteins for the capsid) are replaced by the therapeutic genes.


Herpes simplex virus vectors

Herpes simplex virus 1 (HSV-1) is a DNA virus possessing a double-stranded linear genome of 150 kb. The HSV affords large packaging capacity for insertion of foreign genes. HSV-1 can infect both dividing and nondividing cells. HSV has natural tropism toward neuronal cells, and this property can be exploited for gene therapies for neuronal tumors. HSV-1 particles are relatively stable and can be concentrated to high virus titers, which are valuable for low-volume administration of a large number of viral particles. The virus does not integrate into the host genome, and, therefore, exhibits transient gene expression in infected cells.


Nonviral gene expression system: Plasmid vectors

Unlike viral vectors, which have many inherent risks, such as inflamma-tion and the potential to generate host immune response (both cellular and humoral), plasmid-based nonviral vectors are fairly safe. As illustrated in Figure 26.4, three essential components of gene medicines are a therapeutic gene that encodes a specific therapeutic protein; a gene expression system that recruits host cell machinery to allow the transcription of the encoding gene within a target cell; and a gene delivery system that translocate the expression system to specific location within the body and across the cell membrane barrier.


Figure 26.4 Basic components of a nonviral gene medicine. Therapeutic gene, gene delivery system, and gene expression plasmid are the three basic components of a nonviral gene medicine.

The gene and the gene expression system are the components of plas-mid DNA, which is a circular double-stranded DNA molecule. Basic components of a gene expression plasmid are illustrated in Figure 26.5. Plasmid-based gene expression systems contain a cDNA sequence coding for a therapeutic gene and several other genetic elements, including introns, polyadenylation sequences, and transcript stabilizers to control transcrip-tion, translation, and protein stability. Optional components can be added to an expression plasmid, such as a gene switch, which enables expression of the therapeutic protein to be turned on or off after oral administration of a specific low molecular weight drug. 


Figure 26.5 Basic components of a gene expression plasmid.

The gene delivery system distrib-utes the plasmid to the desired target cells and promotes its internalization into the cells. Once inside the cytoplasm, the plasmid can then translocate to the nucleus, where gene expression begins through the natural cellular processes of transcription and translation.


Gene delivery systems

Plasmid DNA is a long polyanionic polymer. Depending on the number of base pairs, its hydrodynamic size can range from 100 to 200 nm. As the cell membrane is negatively charged, electrostatic repulsion is a bar-rier to the cell membrane translocation of the plasmid DNA. This barrier is commonly overcome through the use of polycationic lipids, polymers, or lipopolymers utilized as gene delivery systems. Most commonly used synthetic gene carriers are cationic polymers and lipids, which condense plasmids into small particles and protect them from degradation by nucle-ases. These positively charged lipids, polymers, or lipopolymers interact with the negatively charged plasmid DNA in aqueous solution to form condensed colloidal particles with low hydrodynamic diameter and an overall positive charge, which have higher cellular uptake. The positively charged gene delivery systems can have interactions with other physiologi-cal proteins that are negatively charged, leading to toxicities. The apparent potency of a plasmid is further reduced by its chemical, enzymatic, and colloidal instability, sequestration by cells of the immune system, uptake and adsorption by nontarget cells and structures, access to target tissues, cellular uptake, and trafficking to the nucleus of the cells. Therefore, there is a growing need for novel delivery systems, which should be safe for repeated administration.

1. Lipid-based gene delivery

Plasmids may be incorporated into cationic or neutral liposomes. With the right selection of lipids for making liposomes, the liposomes can be made pH sensitive so that they are fusogenic (i.e., fuse with the cell membrane) at acidic pH. This feature has been used to facilitate the endosomal disruption and subsequent release of plasmids in the cytoplasm. The cellular uptake process involves incorporation of a plasmid in an intracellular organelle called endosome. The endosome slowly acidifies its contents in an attempt to degrade its contents. The acidified endosome is called a lysosome. The fusogenic lipids fuse with the lysosomal cell membrane as the pH becomes acidic, thus disrupting the lysosome and releasing its cargo. Thus, the plas-mid DNA escapes into the cytoplasm without getting degraded within the lysosome.

Transfection reagent LipofectinTM, for example, is a cationic liposome composed of 1:1 w/w mixture of the cationic lipid N[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA) and the colipid dioleoyl phosphatidylethanolamine (DOPE). Cationic lipids interact elec-trostatically with the negatively charged phosphate backbone of DNA, neu-tralizing the charges and promoting the condensation of DNA into a more compact structure. Usually, cationic lipids are mixed with a zwitterionic or neutral colipid such as DOPE or cholesterol, respectively to form liposomes or micelles. The cationic lipid and colipid are mixed together in chloroform, which is then evaporated to dryness. Water is added to the dried lipid film, and the hydrated film is then either extruded or sonicated to form cationic liposomes. Cationic liposomes have also been prepared by an ethanol injec-tion technique, whereby lipids dissolved in ethanol at a high concentration are injected into an aqueous solution to form liposomes.

As shown in Figure 26.6, the general structure of a cationic lipid has three parts: (1) a hydrophobic lipid anchor group, which helps in forming liposomes (or micellar structures) and can interact with cell membranes; (2) a positively charged headgroup, which interacts with plasmid, leading to its condensation; and (3) a linker group that connects the lipid anchor with the charged headgroup. The net charge of the complex has a signifi-cant effect on transection efficiency (i.e., efficiency of cellular transfer of plasmid DNA) and DNA stability. Usually, positively charged complexes show high transfection efficiency in vitro. The relative proportions of each component and the structure of the head group influence the physicochemi-cal properties of liposome/plasmid complexes.


Figure 26.6 Basic components of a cationic lipid. (a) hydrophobic lipid group, (b) linker group, and (c) cationic headgroup.

2. Peptide-based gene delivery

For site-specific delivery of plasmids, positively charged macromolecules, such as poly(l-lysine) (PLL), histones, protamine, or poly(l-ornithine) may be linked together to a cell-specific ligand and then complexed to plasmids via electrostatic interaction. The resulting complexes retain their ability to interact specifically with target cell receptors, leading to receptor-mediated internaliza-tion of the complex into the cells. Receptor ligands currently being investigated include glycoproteins, transferrin, polymeric immunoglobulin, insulin, epider-mal growth factor (EGF), lectins, folate, malaria circumsporozoite protein, α2-macroglobulin, sugars, integrins (asp-gly-asp [RGD] peptides), thrombo-modulin, surfactant protein A and B, mucin, and the c-kit receptor.

Site-specific gene delivery and expression are influenced by the extent of DNA condensation, the method of complexation, the molecular weights of both polycations and plasmids, and the number of ligand residues bound per polycation molecule. To avoid high cytotoxicity, molecular heteroge-neity, and possible immunogenicity of PLL and polyethylenimine (PEI), molecularly homogenous lysine and arginine-rich peptide-based gene delivery systems are being investigated. Peptides with moieties that pro-vide cooperative hydrophobic behavior of the alkyl chains of cationic lipids would improve the stability of the peptide-based DNA delivery systems. Short synthetic peptides containing the first 23 amino acids of the HA2 subunit of influenza hemagglutinin protein (HA) are attractive because of their pH-dependent lytic properties, with little activity at pH 7 but greater than or equal to a 100-fold increase in transfection efficiency at pH 5.

3. Polymer-based gene delivery

Polymeric biomaterials can be tailored to interact more on cellular and protein levels to achieve high degrees of specificity, activity, and functional-ity. These polymeric materials include (1) polymers that can be covalently attached to proteins and antibodies to form drug conjugates, (2) stimuli sensitive polymers, (3) polymer/cell matrix, (4) functional biodegradable polymers, and (5) polymeric gene carriers. These polymers are being uti-lized for the delivery of proteins, ODNs, and genes. Noncondensing poly-mers, such as polyvinylpyrrolidone (PVP) and pluronics, can also be used for delivery of nucleic acids to muscles and tumors. These polymer-based DNA formulations are hyperosmotic and result in an improved dispersion of plasmids through the extracellular matrix of solid tissues, such as mus-cles or solid tumors, possibly by protecting plasmids from nuclease degra-dation, dispersing plasmids in the muscle, and facilitating their uptake by muscle cells. Synthetic polymers offer a wide array of choices in influencing different aspects of DNA condensation, targeting, cellular uptake, intracel-lular release, and bioactivity.

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