|Year : 2020 | Volume
| Issue : 5 | Page : 341-350
|Gene therapy and its application in dermatology
Tanusree Sarkar1, Somenath Sarkar2, Dwijendra Nath Gangopadhyay3
1 Department of Dermatology, Burdwan Medical College, West Bengal, India
2 Department of Dermatology, B.S Medical College, West Bengal, India
3 Editor, Indian Journal of Dermatology, Kolkata, India
|Date of Web Publication||11-Aug-2020|
Department of Dermatology, B. S. Medical College, Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Gene therapy is an experimental technique to treat genetic diseases. It is based on the introduction of nucleic acid with the help of a vector, into a diseased cell or tissue, to correct the gene expression and thus prevent, halt, or reverse a pathological process. It is a promising treatment approach for genetic diseases, inherited diseases, vaccination, cancer, immunomodulation, as well as healing of some refractory ulcers. Both viral and nonviral vectors can be used to deliver the correct gene. An ideal vector should have the ability for sustained gene expression, acceptable coding capacity, high transduction efficiency, and devoid of mutagenicity. There are different techniques of vector delivery, but these techniques are still under research for assessment of their safety and effectiveness. The major challenges of gene therapy are immunogenicity, mutagenicity, and lack of sustainable therapeutic benefit. Despite these constraints, therapeutic success was obtained in a few genetic and inherited skin diseases. Skin being the largest, superficial, easily accessible and assessable organ of the body, may be a promising target for gene therapy research in the recent future.
Keywords: Dermatology, gene therapy, genetic disease, vector
|How to cite this article:|
Sarkar T, Sarkar S, Gangopadhyay DN. Gene therapy and its application in dermatology. Indian J Dermatol 2020;65:341-50
| Introduction|| |
Genetics plays an important role in several diseases. Some diseases have a definite genetic background whereas some others have genetic factors as an important etiological agent. In some of these diseases, the replacement of the defective gene by a corrected molecule forms a promising line of therapy. Nowadays, advancement in research in genetics has led to an increase in the interest in gene therapy. Gene therapy has been introduced to correct gene-related diseases by replacing the defective genes in these disorders. However, the use of gene therapy is not limited to the field of genodermatoses but also implicated in wound healing, genetic vaccination, cancer treatment, and immunomodulation. This also helps in understanding the pathogenesis of a disease and its prevention.
Gene therapy is defined as the introduction of genetic material or nucleic acid into a cell to repair or compensate the activity of a mutated or abnormal gene or to synthesize beneficial proteins that help to prevent/halt, cure and/or reverse the genetic disease or restore the functional normal protein synthesis.
Gene therapy was first conceptualized in 1972. The first successful gene transfer was shown on a trial in 1990 to treat adenosine deaminase deficiency-severe combined immunodeficiency (ADA-SCID). The first somatic gene therapy was documented for malignant brain tumors by using recombinant DNA technology which makes the tumor cells sensitive to drugs and causes the tumor cells to die.
Gene therapies are of two types, germ-line gene therapy (GGT) and somatic cell gene therapy (SCGT). In GGT, germ cells are modified by introduction of correct/functional genes into their genome. It is heritable and is passed onto the next generation. Application of GGT in human being is still debatable due to insufficient knowledge about possible risks in future, ethical, and technical reasons.
Somatic cell gene therapy introduces a correct/functional gene in any cell other than the germ cell or undifferentiated stem cell. It is not heritable onto the next generation. SCGT is the mainstream for basic research and application of gene therapy to treat gene-related diseases, cancer treatment, vaccination, and wound healing.
Technique of gene therapy
A cell, tissue, or even the whole individual (GGT) may be the target of gene therapy. The most common method of gene therapy is introduction of a correct gene into a nonspecific domain of the genome which replaces a nonfunctional gene. The transformed cell or corrected gene then starts to produce normal protein and restores the healthy phenotype on successive cell division. Gene therapies depend on a few critical elements. For gene therapy, there should be a diseased gene, a therapeutic gene, and an efficient delivery system or carrier called a vector. The vector may be a viral vector (genetically engineered virus) or nonviral vector (naked DNA or DNA complex). Viral vectors are modified in such a way that they can infect the cell to incorporate their gene to host cell chromosomes without creating a disease.
One of the novel approaches of gene therapy is to replace the defective gene through homologous recombination and/or selective reverse mutation to correct the defect and synthesis of normal mRNA. Different techniques of genetic correction include gene restoration, gene augmentation, and gene inhibition. Gene restoration is applied in the case of small point mutation by DNA/RNA chimeric molecule. Gene augmentation is the most common form of gene therapy. It is used to treat a condition where the functional gene is inhibited to produce a functional protein due to mutation. For example, erythropoietin–responsive anemia treated by gene augmentation. Gene inhibition is used to prevent overproduction of some proteins. There are two methods of gene inhibition, antisense nucleic acid (block translation by using antisense oligonucleotide) and ribozyme (down-regulate gene expression by inducing site-specific RNA cleavage). This specific ribozyme used in gene therapy is known as a hammerhead ribozyme and used in gene therapy. There are two types of approaches for delivery of the correct gene, in vivoand ex vivo techniques. In vivotechnique directly delivers the correct gene into the target cell or tissue. The challenge in in vivogene therapy is ensuring specificity and to reach the target cell pinpointly. Genetic material may be introduced directly by injection in a specific tissue, electroporation, gene gun, topical application to normal and wounded surfaces, and bioplastic insertion. In vivogene delivery technique is used where isolation, in vitroculture of the cell and reinsertion of the genetically manipulated cell is not possible. In ex vivo technique defective cells are isolated from the patient, in vitrocell culture is performed and the cultured cells are infected by genetically modified vector for correct gene introduction. The genetically corrected cells are identified and are amplified in vitro; then they are inserted into the host. This technique introduces the correct gene in a correct target cell; as a result, there is less chance of an immune reaction, the higher level of transduction (introduction of foreign DNA in the eukaryotic cell by using a viral vector). But it is specific for those cells only that can be removed from the body.
Gene editing is a type of genetic engineering in which DNA is inserted, deleted, or modified in the genome of a living organism. In gene editing, a tailored nuclease induces a break in DNA double-strand at a targeted site followed by repair with nonhomologous end joining (NHEJ), causing imperfect repair or by homologous recombination (HR) resulting in perfect repair. Two decades ago meganucleases and zinc finger nuclease were used for breaking the DNA, but in recent days, transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) are used for this purpose. This permits gene inactivation through NHEJ repair and correction of disease mutation by HR repair in various animal models much more easily. Significant achievements in gene editing for genetic skin disease have been obtained for dominant dystrophic, junctional, and recessive epidermolysis bullosa.
It is vital to select an appropriate method to deliver DNA. A special molecule has been engineered to increase the efficiency of DNA uptake by the target cells. The molecule used in gene therapy to move recombinant DNA from one cell to another is called a vector. Vector may be viral or nonviral and both of them can be used for in vivoand ex vivo techniques. Success of gene therapy depends on the proper selection of vector.
A number of viruses have been used in human gene therapy, including retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, cytomegalovirus, and vaccinia virus. Among them, retrovirus is the most favorable one. Though the viral vectors have superior efficiency of gene transfer, the main drawback is virus-associated toxicity, complicated manufacturing process, and high cost [Table 1]. Genetic material for viral replication is replaced by the therapeutic gene. Virus carrying therapeutic genes infect the target cell, incorporate the correct gene into the target cell genome, which serves as a template for correct protein synthesis either temporarily or permanently.
Till date retrovirus is the most commonly used vector for cutaneous gene transfer. It is of two types, i) oncoretrovirus, for example, Moloney murine leukemia virus (MMLV), ii) lentivirus originating from human immunodeficiency virus (HIV). Lentivirus has the capability to infect nondividing cells by their ability to deliver pre-integration complex (PIC) across the basement membrane. Epidermal stem cells have low mitotic activity; hence it is more attractive for gene therapy related to dermatology. Oncoretroviruses are efficient for ex vivo
gene delivery. It allows transduction of epidermal stem cells in reconstituted skin tissue, which is essential for long-term transgene (therapeutic gene that has been transferred to another genome) expression. During the process, insertional mutagenesis and silencing of the regulatory gene may lead to malignancy. To prevent this, few techniques are followed, like the incorporation of self-inactivating (SIN) vector which is constructed by introducing a fusion 5' long terminal repeat (LTR) promoter, insertion of sequence like “beta-globin locus,” “control region,” or “Zinc finger nuclease” and introduction of a deletion within U3 region of 3'LTR.
It is a double-stranded DNA virus used as a vector in 23.3% cases worldwide. The main advantages of ADV are its DNA cannot be integrated to host DNA and floats freely in the nucleus, carry a large load of DNA than retrovirus, high transduction efficiency in all types of cells including quiescent cells. So, the viral genome including the therapeutic gene is not transmitted to descendants of target cell, making it unfit to treat a condition where gene therapy is needed for long duration or lifelong. Transgene expression of transduced keratinocytes persist for only 2 weeks, making it ideal for tissue repair and immunotherapy, with less chances of delayed cytotoxicity.
Adeno-Associated virus (AAV)
It is a nonpathogenic parvovirus with single-stranded DNA as genetic material. AAV-2 subtype of the virus has low immunogenicity, stable viral capsid, potential to integrate at specific site, ability to transduce both dividing and quiescent cells like neuron, muscle cell, and eye cells.
Nonviral gene transfer possesses several advantages over viral gene transfer, such as cost-effectivity, low toxicity, low immunogenicity [Table 1]. The disadvantages of these vectors are transient gene expression and low transduction capacity (30%). To improve transduction capacity, gene gun or electroporation technique has developed to increase transduction rate to a higher value (56%). To improve transient gene expression, P-1 based artificial chromosome (PAC) construct has been developed and used to produce stable type VII collagen gene expression in dystrophic epidermolysis bullosa (DEB). It is useful for wound healing and bone regeneration. Uses of nonviral vectors are “loss-of-function” mutation by introducing a plasmid DNA (pDNA) or RNA; “gain-of-function” mutation by RNA interface and micro-RNA. Recent advancement in the field of nonviral gene therapy is to treat monogenic disease by homologous recombination after creating a break in the genome by engineered nuclease. These breaks can be generated by zinc finger nuclease, CRISPR and TALEN.
Naked DNA refers to DNA without any associated histone protein, lipid, or any other molecule that protects it. Naked DNA plasmid and naked DNA-PCR products are also used in gene therapy. In plasmid DNA design, plasmids are propagated in bacterium; hence they have a bacterial replication origin, and a selection marker responsible for antibiotic resistance. A plasmid DNA has tissue-specific promoter, enhancer, splicing introns, locus control region that ensures that the therapeutic gene is adequately expressed in the human target tissue. Inclusion of insulating elements on each end of expression cassette ensures limited influence on other genes and flanking sequence with transposon element allows chromosomal integration of the entire transcription unit, making it safe and efficient. To increase the safety profile, minicircle DNA is developed, that lacks in bacterial backbone sequence, antibiotic resistance gene and this increases the efficiency of transgene expressionin in vivoand ex vivo.,
Plasmid DNA can be delivered by different techniques like topical application, intradermal injection, gene gun (delivering gas, liquid or powder), sonoporation, electroporation, magneto-permeabilization, microneedling, and nano technology. In topical application, plasmid DNA is delivered by aqueous solution. DNA is transferred via hair follicle without affecting the phospholipid rich stratum corneum. Intradermal injection of pDNA incorporates gene mainly into keratinocyte and little into fibroblast and selectively expressed in upper and middle layers of epidermis. Gene gun is needle-free delivery of pDNA driven by helium gas into skin with 10%-20% delivery efficiency, without skin injury. High-pressure flow method, which directs pressurized liquid jet into the skin for immunization, dried-powder vaccine particle accelerates into the skin by epidermal powder immunization. Physical methods are sonoporation in which ultrasound-mediated transient porosities are created in the cell membrane to deliver gene and drug microbubble using sound frequency >20 kHz, electroporation in which long, low voltage pulse is used to surpass the electrical capacity of the cell membrane for transdermal delivery, magneto-permeabilization where the magnetic field is generated for delivery of protein encoded pDNA. Microneedling is minimally invasive epidermal gene transfer, where efficacy is restricted up to the papillary dermis. It is widely used for vaccination. Nanoparticle (NP) is a biodegradable carrier system of variable sizes ranging from 1.5-1000 nm used for gene silencing. Spherical nucleic acid and NP conjugate freely; the conjugate penetrates almost 100% keratinocytes in vitroin mouse epidermis and within hours in the human epidermis after its application.pDNA and minicircle DNA have low transfection (deliberate introduction of naked or purified nucleic acid into eukaryotic cell) efficiency in vivocompared to viral vectors, limiting its effectiveness in gene therapy.
Introduced gene and the viral vector both are foreign proteins for the recipient, hence able to induce an immune reaction. Initially CD4 and CD8 T-cells participate in immune reaction, on successive therapy, memory T-cell also takes part in it and makes the situation worse. Viral vectors stimulate dendritic cells (DC) through type 1 interferons and phosphatidylinositol 3-kinase pathway. Recent researches are focused to find out the way to prevent immune reaction. These are i) tolerogenic DC which are generated with the help of IL-10, NVP347 treatment and ligation of CD45RO/RB; ii) antibody formation against CD45RB and blockade of CD28 and CD40 pathway; iii) suppression of immune system by regulatory T-cell.
Why skin is suitable for gene therapy?
Being superficial, easily manipulatable, observable, and versatile organ, skin is suitable for gene therapy. Dermis is a highly vascular structure with an ability to synthesize and secret a lot of proteins in the circulation. If the skin is transduced with transgene of transferrin, erythropoietin, growth hormone, apolipoprotein E, and factor IX, it secrets the gene product in circulation and its physiological effect can be enormous. Its ability to secret the gene product is further increased by modified progesterone receptor driven transcription. Keratinocyte is the target of choice for gene therapy. It is easily accessible for isolation, in vitrotissue engineering, and can be reconstituted into the subject, in vivoexperimental gene transfer and for monitoring of the modified genome. A study done by Katz et al. reports that keratinocyte secrets 70 proteins into their surroundings. Transduced keratinocytes can synthesize cytokines, like IL-4, IL-6, IL-10, GM-CSF, IFN, monocyte chemotactic and activating factor and secret them into the circulation. Fibroblast, melanocyte, macrophage, endothelial cells are also used as a target of gene therapy.
Gene therapy in Dermatological disorders
Because of the comparative ease of gene transfer, the dermatological disorder is a favorite target of gene therapy. But, to date, the therapy is experimental and not yet in regular use. The autosomal recessive monogenetic disease is easier to treat than an autosomal dominant disease. Other than genetic diseases, skin cancer, chronic wound, and intractable inflammatory disease can also be treated by gene therapy. Candidate skin diseases that can be treated or can be conceptually managed by gene therapy are described below.
Epidermolysis bullosa simplex (EBS)
EBS is a genetically inherited blistering skin disease associated with defective gene expression of the basal keratinocytes. Fifteen genes and 13 proteins are responsible for the specific subtype of this disease. It is caused by a dominant mutation affecting KRT5 and KRT14 keratin genes. Oligonucleotide mediated gene correction technique is used to correct KRT 14 gene. AAV gene-targeting vector with promoter trap design technique was documented beneficial. Antisense technology and RNA interface has also been used. Treatment of EBS patients with heterozygous dominant-negative mutation by silencing or removing the defective allele with the help of short inhibitory RNA (siRNA) and spliceosome-mediated RNA trans-splicing (SMaRT) followed by correct gene insertion is promising but are still experimental.,
Junctional epidermolysis bullosa (JEB)
JEB is of two types, Herlitz JEB (H-JEB) and non-Herlitz JEB (NH-JEB). LAMA3, LAMB3, and LAMC2 genes mainly encode for three chains of laminine 5 α3, β3, and γ2, respectively. LAMB3 gene is mostly affected in H-JEB. MMLV retroviral vector was successfully used for ex vivo correction of LAMB3 gene by using autologous skin grafting in NH-JEB., Nonviral vector phiC31 integrase and transposon were used to correct JEB by cut and paste technique. Dillinger et al. spliced out specific mutant exon to correct collagen 17 mutation of keratinocyte in vitroby using SMaRT in NH-JEB. Laminine-5 and BP-180 deficient H-JEB had been corrected ex vivo by using human skin graft/immune-deficient mouse xenograft model., Nearly 80% clinical correction was achieved with genetically modified skin graft in a patient of JEB.
Dystrophic epidermolysis bullosa (DEB)
DEB is due to mutation in the gene (COL7A1) encoding type VII collagen (C7) and may be of two types, dominant and recessive. Among these two, recessive DEB (RDEB) has dreadful complications, like scarring, esophageal stricture, mitten deformity of hands and feet, and early death due to sepsis or aggressive squamous cell carcinoma., Larger size of the defective gene is the major drawback of gene therapy in DEB. To produce durable, persisting type VII collagen, vector phiC31 bacteriophage integrase along with plasmid expressing COL7A1, plectin cDNA transgene is used. Transgene integration is done into the chromosome of target keratinocytes and fibroblasts of RDEB. Previously it was thought that keratinocyte is responsible for C7 at dermo-epidermal junction (DEJ). But the study done by Woodley et al. shows that it is possible to restore COL7A1 gene expression in RDEB skin by direct intradermal injection of either normal fibroblast or gene-corrected DEB fibroblast in vivo. A study done by Goto et al. showed retroviral gene transfer method for COL7A1 gene into DEB epidermal keratinocytes and dermal fibroblasts, which most efficiently expressed collagen VII at the DEJ. Fibroblast is a better target than keratinocyte, as it produces majority of type VII collagen.Ex vivo gene therapy for inherited skin disorders has been developed in several preclinical studies, with a focus on JEB and RDEB., For ex vivo correction of DEB, human keratinocyte transduced with retrovirus to create transplantable autologous skin on immunocompromised mice. Woodley et al. injected SIN lentiviral vector expressing human C7 in DEB skin grafted onto immunocompromised mice that reverses DEB phenotype for at least 3 months after single intradermal injection. Hence, efficient and long-term transfer of COL7A1 gene in vivois possible by using an engineered lentiviral vector. Local injections of BM-MSCs (bone marrow-derived mesenchymal stromal cells) have shown transient restoration of type VII collagen at the dermal-epidermal junction, an improvement of wound healing, and an increase of skin stability in a hypomorphic RDEB mouse model. Mutation specific antisense oligoribonucleotide (AON) therapy was used to bypass the pathogenic mutation and reverse the DEB phenotype but AON degrade easily after injection into DEB keratinocyte. So, its therapeutic effect is short-lived. Murauer et al. used trans-splicing method by retroviral transduction of keratinocyte with 3'pre trans-splicing molecule to express C7 at the basement membrane. Recently Osborn et al. described gene-specific mutation in RDEB fibroblast by using TALEN novel genome-editing tool. This corrected fibroblast reprogrammed into inducible pleuripotent stem cell that had normal C7 expression. Three “off target” editing events were also documented in addition to “on target” correction of mutation, emphasizing the necessity of establishing the safety profile of such in situ approach. TALEN-mediated correction of a recurrent RDEB mutation was recently achieved by Chamorro et al.
It is a genodermatosis associated with mutation of keratin gene; type 1 disease is due to mutation of gene encoding keratin 6A/KRT16; type 2 disease is due to mutation of gene encoding keratin 6B/KRT17. Leachman et al. used siRNA injection on the affected foot that caused regression of calluses, tenderness without any adverse effect except pain at the site of injection. But the condition reversed after stopping therapy.
It is a genetic disorder due to mutation of the SPINK5 gene responsible for the loss of serine protease inhibitor LEKTI. This causes premature degradation of corneodesmosome and defective keratinization. In vitrotransduction of SPINK5 gene in human keratinocyte by using SIN lentiviral vector to correct expression of LEKTI in human skin graft/murine model was done by Di et al. Roedl et al. used AAV2 mediated viral vector to increase transduction of SPINK5 gene (75%) in wild type keratinocyte to restore LEKTI expression in nonscratch keratinocyte.
Sjogren-Larsson syndrome (SLS)
It develops due to mutation of ALDH3A2 gene, which encodes the enzyme, fatty aldehyde dehydrogenase (FALDH). It is clinically characterized by ichthyosis, mental retardation, and spasticity. Recombinant AAV2 vector is used to transduce SLS keratinocyte, resulting in normal FALDH expression by phenotypically normal keratinocyte.
It is a genodermatosis due to defective nucleotide excision repair (NER) in DNA repair mechanism. Cell without functional NER are prone to UV-induced damage, mutation and development of skin cancer at a younger age (<30 years). MMLV derived retrovirus was used to correct the NER mechanism of keratinocyte DNA repair. On exposure to UV radiation, the corrected keratinocytes continue to repair DNA properly. Corrected keratinocyte holoclones also appear to convey long-term DNA repair while maintaining their DNA repair capabilities. Naldini et al. used a lentiviral vector for transduction into the postmeiotic cells due to its high transduction efficiency and stability. Adenovirus-mediated transduced fibroblast was also used to correct DNA repair XP gene therapy. Plasmid DNA vector should be used in the management of XP, to avoid the risk of insertional mutation.
Lamellar ichthyosis (LI)
It is an autosomal recessive disorder of defective keratinization due to transglutaminase-1 (Tgase-1) deficiency. Keratinocytes of LI patient were transduced in vitrowith an engineered retroviral vector to express correct Tgase-1, which restores defective cross linking of involucrin and filaggrin in immunodeficient mice., This process restores the epidermal barrier function of the immunodeficient mice.
It develops due to steroid sulphatase (STS) deficiency, leading to accumulation of cholesterol sulfate known as “arylsulphatase C.” Gene encoding for STS is completely deleted in 90% of the cases and rest have partial deletion. In vitrotransfection of gene encoding STS causes partial correction of the phenotype with increased cell maturation. Retroviral vector-mediated transduction of STS gene to primary keratinocyte is associated with restoration of full-length STS protein expression and its enzymatic activity. Early loss of transgene expression is more common with plasmid or nonviral naked DNA mediated transfection than retroviral vector-mediated transduction.
Herlequin ichthyosis (HI)
In HI, defect lies in ABCA 12 gene responsible for lipid secretion from lamellar granules of granular layer keratinocyte. Cytomegalovirus based in vitrogene transfer corrects the mutation and restores lipid secretion from lamellar granules.
Bullous congenital ichthyosiform erythroderma (BCIE)/Epidermolytic hyperkeratosis (EH)BCIE caused by mutation of keratin 1(KRT1) and keratin 10(KRT10) genes. It can be corrected by knocking out the mutated allele and expression of the dominant negative protein by using antisense probe. But the main drawback of gene therapy in BCEI is to find out a good antisense probe and varied location of the mutated gene.
It is a disorder of the hematopoietic system. Among various types, erythropoietic protoporphyria (EPP) is characterized by photosensitivity and protoporphyrin deposition in bone marrow, RBC, and other organs due to deficiency of ferrochelatase enzyme. Gene therapy has been tried with SIN lentiviral vector containing human ferrochelatase cDNA driven by human ankyrin-1/β-globin HS-40 chimeric erythroid promoter/enhancer in mice. Ferrochelatase gene was efficiently transferred to erythroid linage of bone marrow and corrected all the clinical and biological alteration.
Recently different clinical trials were undertaken using T-lymphocyte to treat melanoma. Fontana et al. treated melanoma patients with genetically modified autologous lymphocytes expressing the cancer germ-line gene MAGE-A3. Increment in circulating anti-MAGE-A3 antibody was found in 3 out of 10 patients without any toxicity or adverse effect. Dummer et al. gave adenovirus-mediated IL-2 intralesional injection for gene transfer, in patients of advanced solid tumor and metastatic melanoma. Interleukin-2 by stimulating T-cell proliferation induces complete/partial tumor regression. Genetically engineered autologous T-cell was used to express T-cell receptor against NY-ESO-1 antigen to treat metastatic melanoma and metastatic synovial cell carcinoma. Clinical responses were seen in 5 out of 11 patients, with complete regression in 2 patients for at least 1 year. Other clinical trials to treat metastatic melanoma used engineered T-cell receptor against different antigens, like MART-1 and gp100 with variable response.
Squamous cell carcinoma (SCC)
Replication-defective recombinant adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene (”suicide” gene) followed by administration of ganciclovir was used to treat human head and neck SCC successfully in nude mice model.
Wound healing is a complex mechanism with a different sequence of inflammation and has no similarity with genodermatosis. Gene therapy can be applied to wound healing due to i) gene transfer is easier, as there is no epidermal barrier; ii) limited size of area of intervention; iii) therapy is required for a limited time (till healing). Indications of gene therapy are refractory burn wound, diabetic ulcer, vascular ulcer and decubitus ulcer, for rapid healing and to ensure tensile strength of newly formed skin and to avoid complications, like scarring, keloidal changes. Different techniques used for gene therapy are direct application of naked DNA, intraulcer injection, gene-gun delivery, electroporation, microvascular transfection and wound bed implantation. Liechty et al. used adenoviral vector encoding PDGF-β gene for treating ischemia impaired wound healing. Collagen embedded PDGF-β DNA plasmid also promotes wound healing in rabbit. Nonintegrative viral vector are ideal for gene therapy in wound. Diabetic mice who received AAV carrying VEGF-A gene, express increased VEGF-A and showed improved healing than who received AAV-LacZ control. Intradermal injection of plasmid encoded TGF-β gene in diabetic mice, resulted in accelerated cell growth and wound healing. Alogenic keratinocytes are treated ex vivo with plasmid encoded EGF gene, for overexpression of EGF to accelerate wound healing. Nonviral gene transfer of IGF-1 increases granulation synthesis and keratin growth, promotes wound healing in diabetic ulcer of pig. Sonoporation of minicircle VEGF 165 with 1 MHz sound pressure for wound healing in diabetic mice showed reasonable restoration of microarchitecture and wound closure. Perilesional injection of adenovirus encoding PDGF-β in chronic venous leg ulcer showed reduction in size in 1 month. Minicircle plasmid DNA encoding VEGF gene used in combination with cationic dendrimer injected subcutaneously into murine diabetic wound resulting in high level expression of VEGF and wound healing by 6 days. Intradermal injection of sonic hedgehog gene by using biodegradable polynanoparticle in murine full-thickness wound promote expression of VEGF and chemokine stromal cell derived factor-1α significantly accelerate wound healing. Different preclinical studies have been undertaken to assess the role of VEGF, iNOS and EGR-1 genes in vivofor re-epithelialization.
| Conclusion|| |
To make gene therapy clinically available, future research work should focus on both vector design and delivery technique. The real challenges are to increase the effectiveness and durability of nonviral vectors and improve safety, avoid genomic integration and immune reaction associated with viral vectors. As adverse events can be easily monitored on skin it may represent a promising organ to explore these options. The procedure of initiating human gene therapy trials are extremely complex, permissions to be obtained from several authorities and bodies. The cost of any gene therapy trial is huge. In spite of these constrains, new gene therapy is persistently being developed and recently one gene therapy drug is approved in Europe for the treatment of lipoprotein lipase deficiency. We wish to see more gene therapy trials in dermatology and to obtain novel treatment options in different chronic, persistent and genetic dermatological disorders.
Abbreviations other than those described in the text
PCR: Polymerase chain reaction, NVP: N-Vinayl Pyrrolidone, IL: Interleukin, CD: Cluster differentiation, COL: Collagen, KTR: Keratin, SPINK: Serine peptidase inhibitor kazal, LEKTI: Lympho-epithelial kazal type related inhibitor, MAGE: Melanoma associated antigen encoding, PGDF: Platelet-derived growth factor, VEGF: Vascular endothelial growth factor, TGF: Transforming growth factor, EGF: Endothelial growth factor, IGF: Insulin like growth factor, iNOS: Inducible nitric oxide synthase; EGR: Early growth response.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lin M, Pulkkinen L, Uitto J, Yoon K. The gene gun: Current applications in cutaneous gene therapy. Int J Dermatol 2000;39:161-70.
Kay MA. State-of-the-art gene-based therapies: The road ahead. Nat Rev Genet 2011;12:316-28.
Sheridan C. Gene therapy finds its niche. Nat Biotechnol 2011;29:121-8.
Oldfield EH, Ram Z, Culver KW, Blaese MR, DeVroom HL, Anderson WF. Gene therapy for the treatment of brain tumors using intra-tumoral transduction with the thymidine kinase gene and intravenous ganciclovir. Hum Gen Ther 1993;4:39-69.
Lewin A, Glazer PM, Milstone LM. Gene therapy for autosomal dominant disorders of keratin. J Investig Dermatol Symp Proc 2005;10:47-61.
Khavari PA, Rollman O, Vahlquist A. Cutaneous gene transfer for skin and systemic diseases. J Intern Med 2002; 252:1-10.
Baek SC, Lin Q, Robbins PB, Fan H, Khavari PA. Sustainable systemic delivery via a single injection of lentivirus into human skin tissue. Hum Gene Ther 2001;12:1551-8.
Wraight CJ, White PJ. Antisense oligonucleotides in cutaneous therapy. Pharmacol Ther 2001;90:89-104.
Lewin AS, Hauswirth WW. Ribozyme gene therapy: Applications for molecular medicine. Trends Mol Med 2001;7:221-8.
Phylactou LA, Kilpatrick MW, Wood MJ. Ribozymes as therapeutic tools for genetic disease. Hum Mol Genet 1998;7:1649-53.
Robbins PB, Khavari PA. Strategies for cutaneous gene therapy. Curr Probl Dermatol 2000;12:198-203.
Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, et al.
Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 2015;17:233-44.
Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 2014;32:347-55.
Borem A, Santos FR, Bowen DE. Gene therapy. In: Understanding Biotechnology. 1st
ed. New Jersy: Prentice Hall; 2003. p. 87-98.
Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2007—An update. J Gene Med 2007;9:833-42.
Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, et al
. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302:415-9.
Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S. Zinc finger nucleases: Custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucl Acids Res 2005;33:5978-90.
Ginn SL, Alexander IE, Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2012—An update. J Gene Med 2013;15:65-77.
Hirsch T, von Peter S, Dubin G, Mittler D, Jacobsen F, Lehnhardt M, et al.
Adenoviral gene delivery to primary human cutaneous cells and burn wounds. Mol Med 2006;12:199-207.
Chen M, Li W, Fan J, Kasahara N, Woodley D. An efficient gene transduction system for studying gene function in primary human dermal fibroblast and epidermal keratinocytes. Clin Exp Dermatol 2003;28:193-9.
Distler JH, Jungel A, Kurowska-Stolarska M, Michel BA, Gay RE, Gay S, et al
. Nucleofection: A new, highly efficient transfection method for primary human keratinocytes. Exp Dermatol 2005;14:315-20.
Mecklenbeck S, Compton SH, Mejia JE, Cervini R, Hovnanian A, Bruckner-Tuderman L, et al
. A microinjected COL7A1-PAC vector restores synthesis of intact procollagen VII in a dystrohic epidermolysis bullosa keratinocytes cell line. Hum Gene Ther 2002:13:1655-62.
Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science 2003;300:763.
Pabo CO, Peisach E, Grant RA. Design and selection of novel CYS2HIS2 zinc finger proteins. Ann Rev Biochem 2001;70:313-40.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al.
RNA-guided human genome engineering via Cas9. Science 2013;339:823-6.
Osborn MJ, Starker CG, McElroy AN, Webber BR, Riddle MJ, Xia L, et al
. TALEN-based gene correction for epidermolysis bullosa. Mol Ther 2013;21:1151-9.
Tolmachov OE. Building mosaics of therapeutic plasmid gene vectors. Curr Gene Ther 2011;11:466-78.
Darquet AM, Cameron B, Wils P, Scherman D, Crouzet J. A new DNA vehicle for nonviral gene delivery: Supercoiled minicircle. Gene Ther 1997;4:1341-9.
Chen Z. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo
. Mol Ther 2003;8:495-500.
Fan H, Lin Q, Morrissey GR, Khavari PA. Immunization via hair follicles by topical application of naked DNA to normal skin. Nat Biotechnol 1999;17:870-2.
Hengge UR, Chan EF, Foster RA, Walder PS, Vogel JC. Cytokine gene expression in epidermis with biological effects following injection of naked DNA. Nat Gen 1995;10:161-6.
Sawamura D, Meng X, Ina S, Ishikawa H, Tamai K, Nomura K, et al
. In vivo
transfer of a foreign gene to keratinocytes using the hemagglutinating virus of Japan-liposome method. J Invest Dermatol 1997;108:195-9.
Williams RS, Johnston SA, Riedy M, DeVit MJ, McElligott SG, Sanford JC. Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles. Proc Natl Acad Sci 1991;88:2726-30.
Mohammed AJ, AlAwaidy S, Bawikar S, Kurup PJ, Elamir E, Shaban MM, et al.
Fractional doses of inactivated poliovirus vaccine in Oman. N
Engl J Med 2010;362:2351-9.
Dean HJ, Chen D. Epidermal powder immunization against influenza. Vaccine 2004;23:681-6.
Miller DL, Pislaru SV, Greenleaf JE. Sonoporation: Mechanical DNA delivery by ultrasonic cavitation. Somat Cell Mol Genet 2002;27:115-34.
Titomirov AV, Sukharev S, Kistanova E. In vivo
electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim Biophys Acta 1991;1088:131-4.
Kardos TJ, Rabussay DP. Contactless magneto-permeabilization for intracellular plasmid DNA delivery in-vivo
. Hum Vaccin Immunother 2012;8:1707-13.
Pearton M, Saller V, Coulman SA, Gateley C, Anstey AV, Zarnitsyn V, et al.
Microneedle delivery of plasmid DNA to living human skin: Formulation coating, skin insertion and gene expression. J Control Release 2012;160:561-9.
Ditto AJ, Shah PN, Yun YH. Non-viral gene delivery using nanoparticles. Exp Opin Drug Deliv 2009;6:1149-60.
Zheng D, Giljohann DA, Chen DL, Massich MD, Wang XQ, Iordanov H, et al.
Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation. Proc Natl Acad Sci 2012;109:11975-80.
Ghazizadeh S, Kalish RS, Taichman LB. Immune mediated loss of transgene expression in skin: Implication for cutaneous gene therapy. Mol Ther 2003;7:296-303.
Tan PH, Butelspacher SC, Xue SA, Wang YH, Mitchell P, McAlister JC, et al
. Modulation of human dendritic cell function following transduction with viral vectors: Implication for gene therapy. Blood 2005;105:3824-32.
Tan PH, Yates JB, Xue SA, Chan C, Jordan WJ, Harper JE, et al
. Creation of tolerogenic human dendritic cells via intracellular CTLA4: A novel strategy with potential in clinical immunosuppression. Blood 2005;106:2936-43.
Lazarovits AI, Poppema S, Zhang Z, Khandaker M, Le Feuvre CE, Singhal SK, et al
. Prevention and reversal of renal allograft rejection by antibody against CD45RB. Nature 1996;380:717-20.
Larsen CP, Elwood ET, Alexander DZ, Ritchie SC, Hendrix R, Tucker-Burden C, et al
. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 1996;381:434-8.
Tan PH. 9th
American Society of Gene Therapy Annual Meeting. Expert Opin Biol Ther 2006;6:839-42.
Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 1975;6:331-43.
Petersen MJ, Kaplan J, Jorgensen CM, Schmidt LA, Li L, Morgan JR, et al
. Sustained production of human transferrin by transduced fibroblasts implanted into athymic mice: A model for somatic gene therapy. J Invest Dermatol 1995;104:171-6.
Morgan JR, Barrandon Y, Green H, Mulligan RC. Expression of an exogenous growth hormone gene by transplantable human epidermal cells. Science 1987;237:1476-9.
Fenjves ES, Gordon DA, Pershing LK, Williams DL, Taichman LB. Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: Implications for epidermal function and gene therapy. Proc Natl Acad Sci USA 1989;86:8803-7.
Gerrard AJ, Hudson DL, Brownlee GG, Watt FM. Towards gene therapy for haemophilia B using primary human keratinocytes. Nat Genet 1993;3:180-3.
Cao T, Wang XJ, Roop DR. Regulated cutaneous gene delivery: The skin as a bioreactor. Hum Gene Ther 2000;11:2297-300.
Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci 1979;76:5665-8.
Bell E, Ehrlich HP, Buttle DJ, Nakatsuji T. Living tissue formed in vitro
and accepted as skin-equivalent tissue of full thickness. Science 1981;211:1052-4.
Katz AB, Taichman LB. A partial catalogue of proteins secreted by epidermal keratinocytes in culture. J Invest Dermatol 1999;112:818-21.
Meng X, Sawamura D, Ina S, Tamai K, Hanada K, Hashimoto I. Keratinocyte gene therapy: Cytokine gene expression in local keratinocytes and in circulation by introducing cytokine genes into skin. Exp Dermatol 2002;11:456-61.
Greenhalgh DA, Rothnagel JA, Roop DR. Epidermis: An attractive target for gene therapy. J Invest Dermatol 1994;103:63S-9S.
Has C, Spartà G, Kiritsi D, Weibel L, Moeller A, Vega-Warner V, et al
. Integrin α3 mutations with kidney, lung and skin disease. N
Engl J Med 2012;366:1508-14.
Petek L, Fleckman P, Miller D. Efficient KRT14 targeting and functional characterization of transplanted human keratinocytes for the treatment of epidermolysis bullosa simplex. Mol Ther 2010;18:1624-32.
Terron A, McLean WH. Ribozyme gene therapy for keratin disorders. Acta Derm Venereol 2001;81:237.
Wally V, Brunner M, Lettner T, Wagner M, Koller U, Trost A, et al
. K14 mRNA reprogramming for dominant epidermolysis bullosa simplex. Hum Mol Genet 2010;19:4715-25.
Bowden PE. Gene therapy for keratin genodermatoses: Striving forward but obstacles persist. J Invest Dermatol 2011;131:1403-5.
Mavilio F, Pellegrini G, Ferrari S, Di Nunzio F, Di Iorio E, Recchia A, et al
. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 2006;12:1397-402.
Ferrari S, Pellegrini G, Matsui T, Mavilio F, De Luca M. Gene therapy in combination with tissue engineering to treat epidermolysis bullosa. Exp Opin Biol Ther 2006;6:367-78.
Ortiz-Urda S, Lin Q, Yant SR, Keene D, Kay MA, Khavari PA. Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer. Gene Ther 2003;10:1099-104.
Dallinger G, Puttaraju M, Mitchell LG, Yancey KB, Yee C, Klausegger A, et al.
Development of spliceosome-mediated RNA trans
-splicing (SMaRT) for the correction of inherited skin diseases. Exp Dermatol 2003;12:37-46.
Robbins PB, Lin Q, Goodnough JB, Tian H, Chen X, Khavari PA. In vivo
restoration of laminin 5 beta 3 expression and function in junctional epidermolysis bullosa. Proc Natl Acad Sci USA 2001;98:5193-8.
Seitz CS, Giudice GJ, Balding SD, Marinkovich MP, Khavari PA. BP180 gene delivery in junctional epidermolysis bullosa. Gene Ther 1999;6:42-7.
Abbasi J. Clinical advances in human gene therapies. JAMA 2018;319:113-5.
Fine JD, Eady RA, Bauer EA, Briggaman RA, Bruckner-Tuderman L, Christiano A, et al
. Revised classification system for inherited epidermolysis bullosa: Report of the Second International Consensus Meeting on Diagnosis and Classification of Epidermolysis Bullosa. J Am Acad Dermatol 2000;42:1051-66.
Uitto J, Richard G. Progress in epidermolysis bullosa: From eponyms to molecular genetic classification. Clin Dermatol 2005;23:33-40.
Ortiz-Urda S, Thyagarajan B, Keene DR, Lin Q, Fang M, Calos MP, et al
. Stable nonviral genetic correction of inherited human skin disease. Nat Med 2002;8:1166-70.
Ortiz-Urda S, Lin Q, Green C, Keene D, Marinkovich MP, Khavari P. Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue. J Clin Invest 2003;111:251-5.
Woodley DT, Krueger GG, Jorgensen CM, Fairley JA, Atha T, Huang Y, et al
. Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone. J Invest Dermatol 2003;121:1021-8.
Goto M, Sawamura D, Ito K, Abe M, Nishie W, Sakai K, et al
. Fibroblast show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa. J Invest Dermatol 2006;126:766-72.
Abdul-Wahab A, Qasim W, McGrath JA. Gene therapies for inherited skin disorders. Sem Cutan Med Surg 2014;33:83-90.
Uitto J, Bruckner-Tuderman L, Christiano AM, McGrath JA, Has C, South AP, et al.
Progress toward treatment and cure of epidermolysis bullosa: Summary of the DEBRA International Research Symposium EB2015. J Invest Dermatol 2016;136:352-8.
Gache Y, Baldeschi C, Del Rio M, Gagnoux-Palacios L, Larcher F, Lacour JP, et al
. Construction of skin equivalents for gene therapy of recessive dystrophic epidermolysis bullosa. Hum Gene Ther 2004;15:921-33.
Woodley DT, Keene DR, Atha T, Huang Y, Ram R, Kasahara N, et al
. Intradermal injection of lentiviral vectors corrects regenerated human dystrophic epidermolysis bullosa skin tissue in vivo
. Mol Ther 2004;10:318-26.
Khul T, Mezger M, Hausser I, Handgretinger R, Bruckner-Tuderman L, Nystrom A. High local concentrations of intradermal MSCs restore skin integrity and facilitate wound healing in dystrophic epidermolysis bullosa. Mol Ther 2015;23:1368-79.
Goto M, Sawamura D, Nishie W, Sakai K, McMillan JR, Akiyama M, et al
. Targeted skipping of a single exon harboring a premature termination codon mutation: Implications and potential for gene correction therapy for selective dystrophic epidermolysis bullosa patients. J Invest Dermatol 2006;126:2614-20.
Murauer EM, Gache Y, Gratz IK, Klausegger A, Muss W, Gruber C, et al
. Functional correction of type VII collagen expression in dystrophic epidermolysis bullosa. J Invest Dermatol 2011;131:74-83.
Chamorro C, Mencia A, Almarza D, Duarte B, Büning H, Sallach J, et al.
Gene editing for the efficient correction of a recurrent COL7A1 mutation in recessive dystrophic epidermolysis bullosa keratinocytes. Mol Ther Nucleic Acids 2016;5:e307.
Leachman S, Hickerson R, Schwartz M, Bullough E, Hutcherson S, Boucher K, et al
. First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder. Mol Ther 2010;18:442-6.
Di WL, Larcher F, Semenova E, Talbot GE, Harper JI, Del Rio M, et al
. Ex-vivo gene therapy restores LEKTI activity and corrects the architecture of Netherton syndrome-derived skin grafts. Mol Ther 2011;19:408-16.
Roedl D, Oji V, Buters JTM, Behrendt H, Braun-Falco M. rAAV2-mediated restoration of LEKTI in LEKTI-deficient cells from Netherton patients. J Dermatol Sci 2011;61:194-8.
Haug S, Braun Falco M. Adeno-associated virus vectors are able to restore fatty aldehyde dehydrogenase-deficiency. Implications for gene therapy in Sjogren-Larsson syndrome. Arch Dermatol Res 2005;296:568-72.
Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature 2001;411:366-74.
Warrick E, Garcia M, Chagnoleau C, Chevallier O, Bergoglio V, Sartori D, et al
. Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells. Mol Ther 2012;20:798-807.
Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, et al
. In vivo
gene delivery and stable transduction of non-dividing cells by a lentiviral vector. Science 1996;272:263-7.
Marchetto MCN, Muotri A, Burns D, Friedberg E, Menck CFM. Gene transduction in skin cells: Preventing cancer in xeroderma pigmentosum mice. Proc Natl Acad Sci 2004;101:17759-64.
Choate KA, Kinsella TM, Wiliams ML, Nolan GP, Khavari PA. Transglutaminase 1 delivery to lamellar ichthyosis keratinocytes. Hum. Gene Ther 1996;7:2247-53.
Choate KA, Medalie DA, Morgan JR, Khavari PA. Corrective gene transfer in the human skin disorder lamellar ichthyosis. Nat Med 1996;2:1263-7.
Freiberg RA, Choate KA, Deng H, Alperin ES, Shapiro LJ, Khavari PA. A model of corrective gene transfer in X-linked ichthyosis. Hum Mol Genet 1997;6:927-33.
Akiyama M, Sugiyama-Nakagiri Y, Sakai K, McMillan JR, Goto M, Arita K, et al
. Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer. J Clin Invest 2005;115:1777.
Fontana R, Bregni M, Cipponi A, Raccosta L, Rainelli C, Maggioni D, et al
. Peripheral blood lymphocytes genetically modified to express the self/tumor antigen MAGE-A3 induce antitumor immune responses in cancer patients. Blood 2009;113:1651-60.
Dummer R, Rochlitz C, Velu T, Acres B, Limacher JM, Bleuzen P, et al
. Intralesional adenovirus-mediated interleukin-2 gene transfer for advanced solid cancers and melanoma. Mol Ther 2008;16:985-94.
Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, et al.
Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011;29:917-24.
Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, et al.
Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006;314:126-9.
Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC, Hughes MS, et al
. Gene therapy with human and mouse T-cell receptors mediate cancer regression and targets normal tissues expressing cognate antigen. Blood 2009;114:535-46.
O'Malley BW Jr, Chen SH, Schwartz MR, Woo SL. Adenovirus-mediated gene therapy for human head and neck squamous cell cancer in a nude mouse model. Cancer Res 1995;55:1080-5.
Liechty KW, Nesbit M, Herlyn M, Radu A, Adzick NS, Crombleholme TM. Adenoviral-mediated overexpression of platelet-derived growth factor-B corrects ischemic impaired wound healing. J Invest Dermatol 1999;113:375-83.
Tyrone JW, Mogford JE, Chandler LA, Ma C, Xia Y, Pierce GF, et al
. Collagen-embedded platelet-derived growth factor DNA plasmid promotes wound healing in a dermal ulcer model. J Surg Res 2000;93:230-6.
Galeano M, Deodato B, Altavilla D, Cucinotta D, Arsic N, Marini H, et al
. Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse. Diabetologia 2003;46:546-55.
Chesnoy S, Lee P-Y, Huang L. Intradermal injection of transforming growth factor-β1 gene enhances wound healing in genetically diabetic mice. Pharm Res 2003;20:345-50.
Vranckx JJ, Hoeller D, Velander PE, Theopold CF, Petrie N, Takedo A, et al
. Cell suspension cultures of allogenic keratinocytes are efficient carriers for ex vivo
gene transfer and accelerate the healing of full-thickness skin wounds by overexpression of human epidermal growth factor. Wound Repair Regen 2007;15:657-64.
Hirsch T, Spielmann M, Velander P, Zuhaili B, Bleiziffer O, Fossum M, et al
. Insulin-like growth factor-1 gene therapy and cell transplantation in diabetic wounds. J Gene Med 2008;10:1247-52.
Yoon CS, Jung HS, Kwon MJ, Lee SH, Kim CW, Kim MK, et al
. Sonoporation of the minicircle-VEGF165
for wound healing of diabetic mice. Pharm Res 2009;26:794-801.
Margolis D, Morris L, Papadopoulos M, Weinberg L, Filip J, Lang S, et al
. Phase I study of H5.020CMV.PDGF-β to treat venous leg ulcer disease. Mol Ther 2009;17:1822-9.
Kwon MJ, An S, Choi S, Nam K, Jung HS, Yoon CS, et al
. Effective healing of diabetic skin wounds by using nonviral gene therapy based on minicircle vascular endothelial growth factor DNA and a cationic dendrimer. J Gene Med 2012;14:272-8.
Park HJ, Lee J, Kim MJ, Kang T, Jeong Y, Um S, et al
. Sonic hedgehog intradermal gene therapy using a biodegradable poly (β-amino esters) nanoparticle to enhance wound healing. Biomaterials 2012;33:9148-56.
| Article Access Statistics|
| Viewed||819 |
| Printed||22 |
| Emailed||0 |
| PDF Downloaded||110 |
| Comments ||[Add] |