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Targeted Delivery of Genes to Endothelial Cells and Cell‐ and Gene‐Based Therapy in Pulmonary Vascular Diseases

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Abstract

Pulmonary arterial hypertension (PAH) is a devastating disease that, despite significant advances in medical therapies over the last several decades, continues to have an extremely poor prognosis. Gene therapy is a method to deliver therapeutic genes to replace defective or mutant genes or supplement existing cellular processes to modify disease. Over the last few decades, several viral and nonviral methods of gene therapy have been developed for preclinical PAH studies with varying degrees of efficacy. However, these gene delivery methods face challenges of immunogenicity, low transduction rates, and nonspecific targeting which have limited their translation to clinical studies. More recently, the emergence of regenerative approaches using stem and progenitor cells such as endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have offered a new approach to gene therapy. Cell‐based gene therapy is an approach that augments the therapeutic potential of EPCs and MSCs and may deliver on the promise of reversal of established PAH. These new regenerative approaches have shown tremendous potential in preclinical studies; however, large, rigorously designed clinical studies will be necessary to evaluate clinical efficacy and safety. © 2013 American Physiological Society. Compr Physiol 3:1749‐1779, 2013.

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Figure 1. Figure 1. Venn diagram comparing advantages and disadvantages of gene therapy and cell‐based gene therapy.
Figure 2. Figure 2. Mechanisms of entry of viral and nonviral vectors used for gene transfer. Viral vectors: Adenoviruses are the most widely studied viral vectors in gene therapy experiments. Entry of the adenovirus is facilitated by binding of fibers located on the outside surface of the virus to the coxsackie‐adenoviral receptor (CAR). Penton proteins on the surface of the virus are recognized by a cellular integrin, allowing internalization of the adenovirus through an endosome. Acidification of the endosome allows capsid components to dissociate, and for the virion to enter the nucleus where viral dsDNA is released. Retroviruses (or lentiviruses) are single‐stranded RNA viruses, surrounded by an envelope that mediates adhesion to the cell membrane for penetration into cells; however, transfer is restricted to cells possessing the retrovirus‐specific receptor. Following delivery of the gene into the cell, viral RNA is transformed into double‐stranded DNA by a process of reverse transcription. This DNA then integrates randomly into the host cell genome, thereby providing opportunity for prolonged and stable gene expression. Adeno‐associated viruses (AAVs) are nonpathogenic, single‐stranded DNA viruses. Entry of AAVs is facilitated by attachment to the cell surface and recognition of AAV by cell surface receptors such as αVβ5 integrin and FGFR1. Following internalization, the AAV is transported to the nucleus via microtubules where uncoating and release of DNA occurs. AAVs have the ability to integrate stably into the q.13.4‐ter arm of chromosome 19, thereby limiting the risk of insertional mutagenesis. Nonviral Vectors: Plasmid DNA (pDNA) constructs are derived and manufactured in bacteria. pDNA vectors contain the bacterial backbone with key elements such as an antibiotic resistance cassette that are required to produce the vector in bacteria and the gene of interest. Minicircle DNA (mcDNA) improves upon the conventional pDNA by excision of elements of the bacterial backbone that can be immunogenic and by being smaller in size to increase transduction efficiency. Upon reaching the nucleus, the vector remains episomal and is expressed transiently. Transposon DNA contains the gene of interest flanked by terminal inverted repeats (TIRs), which serve as recognition sites for the transposase enzyme. This allows the gene construct to be stably integrated into the host genome by a “cut‐and‐paste” mechanism. These nonviral vectors require a gene delivery method such as DNA complex transfection reagents (lipoplex, polyplex) or electroporation to achieve expression.
Figure 3. Figure 3. Key signaling pathways targeted in gene therapy of PAH. In PAH, homeostasis is mediated by balance of the release of endothelial cell vasodilatory factors such as NO and PGI2 and vasoconstrictors such as thromboxane, and ET‐1 which act on smooth muscle cells to regulate vascular tone. Furthermore, PGI2 and eNOS, and BMP are known to exhibit antiproliferative, proapoptotic effects to regulate smooth muscle growth. Impaired signaling through NO, PGI2 and BMP have been implicated in PAH, which have become targets for gene therapy and cell‐based gene therapy studies through gene transfer of eNOS, PGIS, and BMPR2. In addition, dysregulation of endothelial growth or apoptosis is associated with the development of PAH and may be ameliorated by gene transfer of BMPR2 or VEGFR.
Figure 4. Figure 4. Morphology of early and late outgrowth EPCs using light microscopy. (a) Early outgrowth EPCs with spindle‐like morphology (20× objective). (b) Late outgrowth EPCs with cobblestone morphology and abundant proliferation (20× objective).


Figure 1. Venn diagram comparing advantages and disadvantages of gene therapy and cell‐based gene therapy.


Figure 2. Mechanisms of entry of viral and nonviral vectors used for gene transfer. Viral vectors: Adenoviruses are the most widely studied viral vectors in gene therapy experiments. Entry of the adenovirus is facilitated by binding of fibers located on the outside surface of the virus to the coxsackie‐adenoviral receptor (CAR). Penton proteins on the surface of the virus are recognized by a cellular integrin, allowing internalization of the adenovirus through an endosome. Acidification of the endosome allows capsid components to dissociate, and for the virion to enter the nucleus where viral dsDNA is released. Retroviruses (or lentiviruses) are single‐stranded RNA viruses, surrounded by an envelope that mediates adhesion to the cell membrane for penetration into cells; however, transfer is restricted to cells possessing the retrovirus‐specific receptor. Following delivery of the gene into the cell, viral RNA is transformed into double‐stranded DNA by a process of reverse transcription. This DNA then integrates randomly into the host cell genome, thereby providing opportunity for prolonged and stable gene expression. Adeno‐associated viruses (AAVs) are nonpathogenic, single‐stranded DNA viruses. Entry of AAVs is facilitated by attachment to the cell surface and recognition of AAV by cell surface receptors such as αVβ5 integrin and FGFR1. Following internalization, the AAV is transported to the nucleus via microtubules where uncoating and release of DNA occurs. AAVs have the ability to integrate stably into the q.13.4‐ter arm of chromosome 19, thereby limiting the risk of insertional mutagenesis. Nonviral Vectors: Plasmid DNA (pDNA) constructs are derived and manufactured in bacteria. pDNA vectors contain the bacterial backbone with key elements such as an antibiotic resistance cassette that are required to produce the vector in bacteria and the gene of interest. Minicircle DNA (mcDNA) improves upon the conventional pDNA by excision of elements of the bacterial backbone that can be immunogenic and by being smaller in size to increase transduction efficiency. Upon reaching the nucleus, the vector remains episomal and is expressed transiently. Transposon DNA contains the gene of interest flanked by terminal inverted repeats (TIRs), which serve as recognition sites for the transposase enzyme. This allows the gene construct to be stably integrated into the host genome by a “cut‐and‐paste” mechanism. These nonviral vectors require a gene delivery method such as DNA complex transfection reagents (lipoplex, polyplex) or electroporation to achieve expression.


Figure 3. Key signaling pathways targeted in gene therapy of PAH. In PAH, homeostasis is mediated by balance of the release of endothelial cell vasodilatory factors such as NO and PGI2 and vasoconstrictors such as thromboxane, and ET‐1 which act on smooth muscle cells to regulate vascular tone. Furthermore, PGI2 and eNOS, and BMP are known to exhibit antiproliferative, proapoptotic effects to regulate smooth muscle growth. Impaired signaling through NO, PGI2 and BMP have been implicated in PAH, which have become targets for gene therapy and cell‐based gene therapy studies through gene transfer of eNOS, PGIS, and BMPR2. In addition, dysregulation of endothelial growth or apoptosis is associated with the development of PAH and may be ameliorated by gene transfer of BMPR2 or VEGFR.


Figure 4. Morphology of early and late outgrowth EPCs using light microscopy. (a) Early outgrowth EPCs with spindle‐like morphology (20× objective). (b) Late outgrowth EPCs with cobblestone morphology and abundant proliferation (20× objective).
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Colin M. Suen, Shirley H.J. Mei, Lakshmi Kugathasan, Duncan J. Stewart. Targeted Delivery of Genes to Endothelial Cells and Cell‐ and Gene‐Based Therapy in Pulmonary Vascular Diseases. Compr Physiol 2013, 3: 1749-1779. doi: 10.1002/cphy.c120034