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Targeted Gene Delivery: Future of High Efficiency Gene Therapy
#1
This is post no. 1 under the main topic.
Since the day, Ashanti Desilva, a four year old little girl from United States, suffering from ADA SCID (an immune deficiency disease) was operated by the first ever gene therapy attempt in the medical history in 1990, a lot has happened in the field of Gene Therapy. Speculations were always rife about the fatal consequences of Gene Therapy (alteration of normal genetic make-up, physiological rejection etc), but they could never out-weigh the foreseen advantages of permanent cure of diseases as deadly as Cancer.

Our physiology is an outcome of the housekeeping genes, or in better words, our normal physiology is an outcome of the normal functioning of the housekeeping genes expressing round the clock in our system (body). Whereas, the house keeping genes ensure the optimal functioning of metabolic pathways ranging from DNA synthesis to food digestion, there are another set of genes called oncogenes (cancer causing genes) whose dormancy is extremely crucial for the normal functioning of the system. In dormant (or inactive) state, these oncogenes are termed proto-oncogenes and are unable to exert their effect i.e uncontrolled cell division. And, any mutation in either the house keeping genes or proto-oncogenes, can severely harm the normal functioning of the physiological system, manifested in the form of diseases like Thalassemia, Sickle Cell Anemia and numerous forms of cancers etc. This is where gene therapy pronounces itself as the most efficient and lasting cure for such fatal diseases; because it’s always best to remove the cause than to treat the symptoms.

The basic philosophy of the gene therapy is to replace the mal-functioning/mutated gene with a normal gene. In words, it might seem as simple as swapping an old ball with a new one, but in practice gene swapping is the most ambitious treatment for any disease, whose chances of success are as low as tracing a needle in the haystack! A successful gene therapy has numerous check lists to follow, missing a single pre-requisite can mar the attempt and rather pose threat to the subject. One of the most important requirement in gene therapy is targeted delivery of the gene(s) to the genome of the cell(s), it’s intended to swap genes with. As in the case of ADA SCID of Ashanthi, one of the targeted approach is to transfer the required gene into the target cells by taking them out of the body and them conducting an in-vitro transfer to the cultured cells. This approach, called ex-vivo, requires extraction of the desired cell(s) from the body, a high class culturing facility for the cells and then a mechanism for regular injection of those cells in the body at the extracted location, making it inherently cumbrous. Other method, the in-vivo mode of gene delivery, involves direct injection of the transgene into the body through various routes, with an aim of site-specific delivery, integration and expression of the gene of interest. Now, the term direct injection shouldn't be misinterpreted, as the DNA/transgene cannot be injected as such in naked form. A naked DNA may be treated as an antigen and defense response may be initiated by the body to wipe the antigen. Apart from that, various nucleases present in the body fluids/cells may degrade the naked DNA much before its anticipated delivery to the target site.

Modes of In-vivo Targeted Gene Delivery

The in-vivo procedure can utilize two modes of delivering the transgene to the target site:
a. Viral Mode
b. Non-Viral Mode

Viral mode exploits the ability of viral vectors to integrate their DNA into the host genome, and replicating with the host thereafter. This is one of the most efficient mode of gene transfer in terms of the probability of successful gene integration and expression. But the concern over the use of Viral vectors in Humans has always been a roadblock in this regard.

Considering the limitation of viral vectors, numerous attempts have been made in developing an efficient non-viral mode of gene delivery. Use of gene gun, polyplexes and lipoplexes, are some of the conventionally tried methods to deliver genes into the cells. But considering the stringent requirement of the gene therapy, the rate of success with such physical methods is very low. It is equally probable that the gene carrying complex may reach at an unintended site and integrate the DNA in the normal cells causing adverse side effects. The obvious challenge in using these physical means of delivery lies in targeted delivery of the delivery complex. In order to achieve a site specific delivery, tagging of these complexes with some ligands complementary to the surface antigens of the targeted cells is the most common and efficient approach. Infact, ligand tagged nanoparticles have emerged as the complexes of choice for delivering the genes to the target site. For example, Tissue Factor (TF) expressed by injured cells of the body has become an address of choice for nanoparticle mediated drug and gene delivery to the injured tissue inside the body. EGFP-EGF1 is tagged on the nanoparticles which owing to it’s affinity towards TF directs the nanoparticles carrying the drug/gene payload to the injured site. The nature of nanoparticles under use in most cases is PLGA or poly(lactic-co-glycolic acid), which exhibits extraordinary biocompatibility and biodegradability. In a recent publication by Department of Surgery, Guangdong Provincial Stomatological Hospital, Southern Medical University, Guangzhou, People's Republic of China, use of Quantum Dots (QDs) as vectors for targeted survivin gene siRNA delivery was reported. Use of QDs enables real time probing of the successful gene delivery and it’s expression levels. For those who are unaware of the concept of QDs, these are tiny nano-particles with a size range of 2-10nm and are chemically selenides of cadmium or zinc. Their extra-ordinary small size enables unique electrical and optical properties, which can be studied in the form of photonic emissions.

Evidently, a lot of progress has taken place in the field of targeted gene delivery for an efficient gene therapy. New approaches like ligand coated Nanoparticles and use of Quantum Dots exhibit some of the big achievements in this field in a period as short as just 2 decades. With the widening scope of targeted delivery, diseases like advanced Cancer and even HIV have been reportedly cured in some instances. And, the scope will keep expanding it’s horizons with increasing knowledge of genetic behavior of diseases and newer means of delivering the medicinal gene to the diseased site.
 
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#2
This is post no. 2 under the main topic.
Targeted gene delivery and Cystic Fibrosis

The previous article eloquently highlights the advances in targeting gene delivery over the last couple of decades. Part of the challenge of targeting gene delivery lies in the individual cell and tissue environments which need to be targeted in different diseases. Recent studies have addressed the issue of target gene delivery to the lung for potentially targeted treatment of diseases such as Cystic Fibrosis (CF). CF is a devastating autosomal recessively inherited genetic disease that affects most notably the lungs, but also the liver, pancreas and intestines. It involves a defect in epithelial transport of sodium and chlorine and is caused by mutations in the CFTR gene. The aim of gene therapy in CF would be to introduce a normal copy of the CFTR gene into cells. However, the field has been fraught with difficulties including poor vector take-up by cell and problems with humoral immunity associated with adenoviral vectors.

However, there have been some promising results. In a study from the Univeristy of Florida, a CFTR knockout mouse model of a IgE-mediated hypersensitivity (allergic bronchopulmonary aspergillosis (ABPA))syndrome which occurs in approximately 15% of CF patients was studied. A truncated CFTR was delivered to mouse airway epiltelium via intra-tracheal (IT) delivery of recombinant adeno-associated virus and was found to attenuate the hyper-IgE response. However, more recent studies have focused on non-viral gene delivery systems such as the nanoparticles referred to in the previous article. Chitosan is a polysaccharide whose properties have led to its use drug delivery systems.Guanidinylated chitosan (GCS) has been developed as a non-viral vector termed the chitosan-derived nanodelivery vehicle (SGCS), that can be used to deliver nanoparticles to lung cells. This was recently shown to successfully deliver siRNA to lung cells. In another example, chitosan-DNA-FAP-B nanoparticles were identified as novel non-viral vectors for specific gene delivery to lung epithelial cells. They were effective in targeted gene delivery to fibronectin molecules of lung epithelial cell membrane and can be delivered by aerosol to give several-fold greater gene expression in mice lungs compared with Chitosan-DNA nanoparticles. Chitosan-DNA nanoparticles can also be effectively delivered to liver cells in mice, which has further relevance for CF.

Studies on naonparticle delivery are focusing on factors such as cytotoxicity and effectiveness of delivery via aerosol. The chitosan-based vectors may be a promising avenue for further development in gene therapy for diseases such as CF.

Sources

DAI, H. et al., 2006. Chitosan-DNA nanoparticles delivered by intrabiliary infusion enhance liver-targeted gene delivery. International Journal Of Nanomedicine, 1(4), pp. 507-522

FLOTTE, T.R. and LAUBE, B.L., 2001. Gene therapy in cystic fibrosis. Chest, 120(3), pp. 124S-131S

LUO, Y. et al., 2012. An inhalable ß2-adrenoceptor ligand-directed guanidinylated chitosan carrier for targeted delivery of siRNA to lung. Journal Of Controlled Release: Official Journal Of The Controlled Release Society, 162(1), pp. 28-36

MOHAMMADI, Z. et al., 2011. In vivo transfection study of chitosan-DNA-FAP-B nanoparticles as a new non viral vector for gene delivery to the lung. International journal of pharmaceutics, 421(1), pp. 183-188

MOHAMMADI, Z. et al., 2012. Stability studies of chitosan-DNA-FAP-B nanoparticles for gene delivery to lung epithelial cells. Acta Pharmaceutica (Zagreb, Croatia), 62(1), pp. 83-92

MUELLER, C. et al., 2008. Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene. The journal of gene medicine, 10(1), pp. 51-60

YEO, Y., 2010. Battling with environments: drug delivery to target tissues with particles and functional biomaterials. Therapeutic Delivery, 1(6), pp. 757-761
 
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#3
This is post no. 3 under the main topic.
Targeted Gene Therapy in Cancer:

In recent past the thought of using gene therapy as a modality in the handling of diseases other than genetically inherited, a monogenic disorder has taken origin. This is mainly understandable in the field of oncology where currently more than 100 clinical trials have been approved worldwide. Some of the exciting progress that has recently been made with admiration to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing regular tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the competent delivery of DNA to tumor sites remains a alarming task, development has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be joint in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). On the other hand, expression could be regulated outwardly with the use of radiation-induced promoters or tetracycline-responsive elements. One more novel possibility that will be discussed is the guideline of therapeutic gene products by tumor-specific gene splicing. Gene expression could also be targeted at circumstances specific to the tumor microenvironment, such as glucose deprivation and hypoxia. It has concentrated on hypoxia-targeted gene expression. Chronic hypoxia happens in tissue that is more than 100-200 microns away from a functional blood supply. In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is jumbled. Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 2.5 mmHg), readings not seen in usual tissue. This is a most important problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy. However, severe hypoxia is also a physiological condition specific to tumors, which constructs it a potentially exploitable target. People have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to manage gene expression in human tumor cells in vitro and in experimental tumors. Other imaginative strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the make use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes).
 
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#4
This is post no. 4 under the main topic.
Our physiology is an outcome of the housekeeping genes, or in better words, our normal physiology is an outcome of the normal functioning of the housekeeping genes expressing round the clock in our system (body). Whereas, the house keeping genes ensure the optimal functioning of metabolic pathways ranging from DNA synthesis to food digestion, there are another set of genes called oncogenes (cancer causing genes) whose dormancy is extremely crucial for the normal functioning of the system. In dormant (or inactive) state, these oncogenes are termed proto-oncogenes and are unable to exert their effect i.e uncontrolled cell division. And, any mutation in either the house keeping genes or proto-oncogenes, can severely harm the normal functioning of the physiological system, manifested in the form of diseases like Thalassemia, Sickle Cell Anemia and numerous forms of cancers etc. This is where gene therapy pronounces itself as the most efficient and lasting cure for such fatal diseases; because it’s always best to remove the cause than to treat the symptoms.
 
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#5
This is post no. 5 under the main topic.
In dormant (or inactive) state, these oncogenes are termed proto-oncogenes and are unable to exert their effect i.e uncontrolled cell division
 
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