A review of the literature on the use of CRISPR/Cas9 gene therapy to treat hepatocellular carcinoma

Noncoding RNAs instruct the Cas9 nuclease to site speifillyl cleave DNA in the CRISPR/Cas9 system.Despite the high incidence of hepatocellular carcinoma(HCC),the patient's outcome is poor.As a result of the emergence of therapeutic resistance in HCC patients,dlinicians have faced difficulties in treating such tumor.In addition,CRISPR/Cas9 screens were used to identify genes that improve the dlinical response of HCC patients.It is the objective of this article to summarize the current understanding of the use of the CRISPR/Cas9 system for the treatment of cancer,with a particular emphasis on HCC as part of the current state of knowledge.Thus,in order to locate recent developments in oncology research,we examined both the Scopus database and the PubMed database.The ability to selectively interfere with gene expression in combinatorial CRISPR/Cas9 screening can lead to the discovery of new effective HCC treatment regimens by combining clinically approved drugs.Drug resistance can be overcome with the help of the CRISPR/Cas9 system.HCC signature genes and resistance to treatment have been uncovered by genome-scale CRISPR activation screening although this method is not without limitations.It has been extensively examined whether CRISPR can be used as a tool for disease research and gene therapy.CRISPR and its applications to tumor research,particularly in HCC,are examined in this study through a review of the literature.

The concept of gene therapy for glaucoma:the dream that has not come true yet

Gene therapies,despite of being a relatively new therapeutic approach,have a potential to become an important alternative to current treatment strategies in glaucoma.Since glaucoma is not considered a single gene disease,the identified goals of gene therapy would be rather to provide neuroprotection of retinal ganglion cells,especially,in intraocular-pressure-independent manner.The most commonly reported type of vector for gene delivery in glaucoma studies is adeno-associated virus serotype 2 that has a high tro pism to retinal ganglion cells,res ulting in long-term expression and low immunogenic profile.The gene thera py studies recruit inducible and genetic animal models of optic neuropathy,like DBA/2J mice model of high-tension glaucoma and the optic nerve crush-model.Reported gene therapy-based neuroprotection of retinal ganglion cells is targeting specific genes translating to growth factors(i.e.,brain derived neurotrophic factor,and its receptor TrkB),regulation of apoptosis and neurodegeneration(i.e.,Bcl-xl,Xiap,FAS system,nicotinamide mononucleotide adenylyl transferase 2,Digit3 and Sarm1),immunomodulation(i.e.,Crry,C3 complement),modulation of neuroinflammation(i.e.,e rythropoietin),reduction of excitotoxicity(i.e.,Com KIlα)and transcription regulation(i.e.,Max,Nrf2).On the other hand,some of gene therapy studies focus on lowering intra ocular pressure,by impacting genes involved in both,decreasing aqueous humor production(i.e.,aquaporin 1),and increasing outflow facility(i.e.,COX2,prostaglandin F2a receptor,RhoA/RhoA kinase signaling pathway,MMP1,Myocilin).The goal of this review is to summarize the current stateof-art and the direction of development of gene therapy strategies for glaucomatous neuropathy.

Facing ethical concerns in the age of precise gene therapy:Outlook on inherited arrhythmias

This editorial,comments on the article by Spartalis et al published in the recent issue of the World Journal of Cardiology.We here provide an outlook on potential ethical concerns related to the future application of gene therapy in the field of inherited arrhythmias.As monogenic diseases with no or few therapeutic options available through standard care,inherited arrhythmias are ideal candidates to gene therapy in their treatment.Patients with inherited arrhythmias typically have a poor quality of life,especially young people engaged in agonistic sports.While genome editing for treatment of inherited arrhythmias still has theoretical application,advances in CRISPR/Cas9 technology now allows the generation of knock-in animal models of the disease.However,clinical translation is somehow expected soon and this make consistent discussing about ethical concerns related to gene editing in inherited arrhythmias.Genomic off-target activity is a known technical issue,but its relationship with ethnical and individual genetical diversity raises concerns about an equitable accessibility.Meanwhile,the costeffectiveness may further limit an equal distribution of gene therapies.The economic burden of gene therapies on healthcare systems is is increasingly recognized as a pressing concern.A growing body of studies are reporting uncertainty in payback periods with intuitive short-term effects for insurance-based healthcare systems,but potential concerns for universal healthcare systems in the long term as well.Altogether,those aspects strongly indicate a need of regulatory entities to manage those issues.

Presymptomatic Diagnosis and Gene Therapy for Alzheimer’s Disease: Genomic, Therapeutic, and Ethical Aspects—A Systematic Review

Over the past three decades, genomic and epigenetic sciences have identified more than 70 genes involved in the molecular pathophysiology of Alzheimer’s disease (AD). DNA methylation, abnormal histone and chromatin regulation and the action of various miRNAs induce AD. The identification of mutated genes has paved the way for the development of diagnostic kits and the initiation of gene therapy trials. However, despite major advances in neuroscience research, there is yet no suitable treatment for AD. Therefore, the early diagnosis of this neurodegenerative disease raises several ethical questions, including the balance between the principle of non-maleficence and the principle of beneficence. The aims of this research were to present the genomic and ethical aspects of AD, and to highlight the ethical principles involved in its presymptomatic diagnosis and therapy. A systematic review of the literature in PubMed, Google Scholar and Science Direct was carried out to outline the genomic aspects and ethical principles relating not only to the presymptomatic diagnosis of AD, but also to its gene therapy. A total of 16 publications were selected. AD is a multifactorial disease that can be genetically classified into Sporadic Alzheimer’s Disease and Familial Alzheimer’s Disease based on family history. Gene therapy targeting specific disease-causing genes is a promising therapeutic strategy. Advancements in artificial intelligence applications may enable the prediction of AD onset several years in advance. While early diagnosis of AD may empower patients with full decision competence for early decision-making, it also carries implications for the patient’s family members, who are at risk of developing the disease, potentially becoming a source of confusion or anxiety. AD has a significant impact on the life of individuals at risk and their families. Given the absence of disease modifying therapy, genetic screening and early diagnosis for this condition raise ethical issues that must be carefully considered in the context of fundamental bioethical principles, including autonomy, beneficence, non-maleficence, and justice.

Gene therapy for Parkinson’s Disease and Ethical Challenges: A Systematic Review

Background: Parkinson’s disease (PD) is a complex, multifactorial neurodegenerative disorder with a pathophysiology deriving from the synergy of abnormal aggregation of neuroinflammation, synuclein and dysfunction of lysosomes, mitochondria and synaptic transport difficulties influenced by genetic and idiopathic factors. Worldwide, PD has a prevalence of 2-3% in people over the age of 65. To date, there is no certified, effective treatment for PD. Aim: The aims of this research were: (i) to present, on the basis of recent advances in molecular genetics and epigenetics, the genomic aspects and challenges of gene therapy trials for PD;(ii) to outline the ethical principles applicable to therapeutic trials for PD. Method: A systematic literature review was carried out to identify relevant articles reporting on genomic aspects and gene therapy in PD from 2001 to October 2023. The search was conducted in French and/or English in three databases: PubMed, Google Scholar and Science Direct. PRISMA guidelines were used in this systematic review. Results: A total of thirty-three publications were selected. An inductive thematic analysis revealed that numerous genetic mutations (SNCA, Parkin, PINK1, DJ-1, LRRK2, ATP13A2, VPS35, Parkin/PRKN, PINK1, DJ1/PARK7) and epigenetic events such as the action of certain miRNAs (miR-7, miR-153, miR-133b, miR-124, miR-137) are responsible for the onset of PD, and that genetic therapy for this pathology raises ethical questions that need to be elucidated in the light of the bioethical principles of autonomy, beneficence, non-maleficence and justice. Conclusion: There is no zero risk in biotechnology. Then, it will be necessary to assess all the potential risks of Parkinson disease’s gene therapy to make the right decision. It is therefore essential to pursue research and, with the guidance of ethics, to advance treatment options and meet the challenges of brain manipulation and its impact on human identity. The golden rule of medicine remains: “Primum non nocere”.

Nanotechnology-based gene therapy as a credible tool in the treatment of Alzheimer’s disease

Toxic aggregated amyloid-βaccumulation is a key pathogenic event in Alzheimer’s disease.Treatment approaches have focused on the suppression,deferral,or dispersion of amyloid-βfibers and plaques.Gene therapy has evolved as a potential therapeutic option for treating Alzheimer’s disease,owing to its rapid advancement over the recent decade.Small interfering ribonucleic acid has recently garnered considerable attention in gene therapy owing to its ability to down-regulate genes with high sequence specificity and an almost limitless number of therapeutic targets,including those that were once considered undruggable.However,lackluster cellular uptake and the destabilization of small interfering ribonucleic acid in its biological environment restrict its therapeutic application,necessitating the development of a vector that can safeguard the genetic material from early destruction within the bloodstream while effectively delivering therapeutic genes across the bloodbrain barrier.Nanotechnology has emerged as a possible solution,and several delivery systems utilizing nanoparticles have been shown to bypass key challenges regarding small interfering ribonucleic acid delivery.By reducing the enzymatic breakdown of genetic components,nanomaterials as gene carriers have considerably enhanced the efficiency of gene therapy.Liposomes,polymeric nanoparticles,magnetic nanoparticles,dendrimers,and micelles are examples of nanocarriers that have been designed,and each has its own set of features.Furthermore,recent advances in the specific delivery of neurotrophic compounds via gene therapy have provided promising results in relation to augmenting cognitive abilities.In this paper,we highlight the use of different nanocarriers in targeted gene delivery and small interfering ribonucleic acid-mediated gene silencing as a potential platform for treating Alzheimer’s disease.

Brain and spinal cord trauma:what we know about the therapeutic potential of insulin growth factor 1 gene therapy

Although little attention has been paid to cognitive and emotional dysfunctions observed in patients after spinal co rd injury,several reports have described impairments in cognitive abilities.Our group also has contributed significantly to the study of cognitive impairments in a rat model of spinal co rd injury.These findings are very significant because they demonstrate that cognitive and mood deficits are not induced by lifestyle changes,drugs of abuse,and combined medication.They are related to changes in brain structures involved in cognition and emotion,such as the hippocampus.Chronic spinal cord injury decreases neurogenesis,enhances glial reactivity leading to hippocampal neuroinflammation,and trigge rs cognitive deficits.These brain distal abnormalities are recently called te rtiary damage.Given that there is no treatment for Tertiary Damage,insulin growth factor 1 gene therapy emerges as a good candidate.Insulin growth factor 1 gene thera py recove rs neurogenesis and induces the polarization from pro-inflammato ry towards anti-inflammatory microglial phenotypes,which represents a potential strategy to treat the neuroinflammation that supports te rtiary damage.Insulin growth factor 1 gene therapy can be extended to other central nervous system pathologies such as traumatic brain injury where the neuroinflammatory component is crucial.Insulin growth factor 1 gene therapy could emerge as a new therapeutic strategy for treating traumatic brain injury and spinal cord injury.

CMT1A current gene therapy approaches and promising biomarkers

Charcot-Marie-Tooth neuropathies(CMT)constitute a group of common but highly heterogeneous,non-syndromic genetic disorders affecting predominantly the peripheral nervous system.CMT type 1A(CMT1A)is the most frequent type and accounts for almost~50%of all diagnosed CMT cases.CMT1A results from the duplication of the peripheral myelin protein 22(PMP22)gene.Overexpression of PMP22 protein overloads the protein folding apparatus in Schwann cells and activates the unfolded protein response.This leads to Schwann cell apoptosis,dys-and de-myelination and secondary axonal degeneration,ultimately causing neurological disabilities.During the last decades,several different gene therapies have been developed to treat CMT1A.Almost all of them remain at the pre-clinical stage using CMT1A animal models overexpressing PMP22.The therapeutic goal is to achieve gene silencing,directly or indirectly,thereby reversing the CMT1A genetic mechanism allowing the recovery of myelination and prevention of axonal loss.As promising treatments are rapidly emerging,treatment-responsive and clinically relevant biomarkers are becoming necessary.These biomarkers and sensitive clinical evaluation tools will facilitate the design and successful completion of future clinical trials for CMT1A.

Ionizable drug delivery systems for efficient and selective gene therapy

Gene therapy has shown great potential to treat various diseases by repairing the abnormal gene function.However,a great challenge in bringing the nucleic acid formulations to the market is the safe and effective delivery to the specific tissues and cells.To be excited,the development of ionizable drug delivery systems(IDDSs)has promoted a great breakthrough as evidenced by the approval of the BNT162b2 vaccine for prevention of coronavirus disease 2019(COVID-19)in 2021.Compared with conventional cationic gene vectors,IDDSs can decrease the toxicity of carriers to cell membranes,and increase cellular uptake and endosomal escape of nucleic acids by their unique pH-responsive structures.Despite the progress,there remain necessary requirements for designing more efficient IDDSs for precise gene therapy.Herein,we systematically classify the IDDSs and summarize the characteristics and advantages of IDDSs in order to explore the underlying design mechanisms.The delivery mechanisms and therapeutic applications of IDDSs are comprehensively reviewed for the delivery of plasmid DNA(pDNA)and four kinds of RNA.In particular,organ selecting considerations and high-throughput screening are highlighted to explore efficiently multifunctional ionizable nanomaterials with superior gene delivery capacity.We anticipate providing references for researchers to rationally design more efficient and accurate targeted gene delivery systems in the future,and indicate ideas for developing next generation gene vectors.

Bioorthogonal Engineered Virus‑Like Nanoparticles for Efficient Gene Therapy

Gene therapy offers potentially transformative strategies for major human diseases.However,one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue.Here,we report a novel virus-like nanoparticle,the bioorthgonal engineered viruslike recombinant biosome(reBiosome),for efficient gene therapies of cancer and inflammatory diseases.The mutant virus-like biosome(mBiosome)is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site,and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry.The results show that the reBiosome exhibits reduced virus-like immunogenicity,prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci(like tumor and arthritic tissue).Furthermore,reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems,respectively.In conclusion,this study develops a universal,safe and efficient platform for gene therapies for cancer and inflammatory diseases.

Gene Therapy Activates Retinal Pigment Epithelium Cell Proliferation for Age-related Macular Degeneration in a Mouse Model

Objective Age-related macular degeneration(AMD)is a degenerative retinal disease.The degeneration or death of retinal pigment epithelium(RPE)cells is implicated in the pathogenesis of AMD.This study aimed to activate the proliferation of RPE cells in vivo by using an adeno-associated virus(AAV)vector encodingβ-catenin to treat AMD in a mouse model.Methods Mice were intravitreally injected with AAV2/8-Y733F-VMD2-β-catenin for 2 or 4 weeks,andβ-catenin expression was measured using immunofluorescence staining,real-time quantitative reverse transcription polymerase chain reaction(PCR),and Western blotting.The function ofβ-catenin was determined using retinal flat mounts and laser-induced damage models.Finally,the safety of AAV2/8-Y733F-VMD2-β-catenin was evaluated by multiple intravitreal injections.Results AAV2/8-Y733F-VMD2-β-catenin induced the expression ofβ-catenin in RPE cells.It activated the proliferation of RPE cells and increased cyclin D1 expression.It was beneficial to the recovery of laser-induced damage by activating the proliferation of RPE cells.Furthermore,it could induce apoptosis of RPE cells by increasing the expression of Trp53,Bax and caspase3 while decreasing the expression of Bcl-2.Conclusion AAV2/8-Y733F-VMD2-β-catenin increasedβ-catenin expression in RPE cells,activated RPE cell proliferation,and helped mice heal from laser-induced eye injury.Furthermore,it could induce the apoptosis of RPE cells.Therefore,it may be a safe approach for AMD treatment.

Construction of Bifidobacterium Infantis/CD Targeting Gene Therapy System

Objective： To construct Bifidobacterium Infantis/CD targeting gene therapy system. Methods： CD gene was amplified from E. Coli K12λ using PCR method, pGEX-1LamdaT plasmid and CD gene were digested with dual restriction endonucleas of EcoR Ⅰ and BamH Ⅰ and two segments of 4.9 kb and 1.3 kb were obtained. T4 DNA ligase was added to these two segments to make a recombinant CD/pGEX-1LamdaT plasmid. Then the recombinant plasmid was transfected into Bifidobacterium Infantis by electroporation. The recombinant plasmid was extracted from the positively transfected Bifidobacterium Infantis and digested with dual restriction endonucleases. Then the size of digested fragments was detected and sequencing of the gene segment inserted in extracted recombinant plasmid was performed according to the method of Sanger dideoxynucleotide triphosphate chain termination. Results： 6.2 kb recombinant plasmid was obtained from the positively transfected bacterial colony of Bifidobacterium Infantis. After being digested with dual restriction endonucleases, two segments of approximate 4.9 kb and 1.3 kb were gained from the extracted recombinant plasmid, which were equal to the size of pGEX-1LamdaT plasmid and CD gene, respectively. The full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. Conclusion： The foreign gene, CD gene was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium Infantis. Bifidobacterium Infantis/CD targeting gene therapy system was successfully constructed.

Advances in gene therapy of liver cirrhosis: a review

INTRODUCTIONLiver fibrosis or cirrhosis is a common progressively pathological lesion of chronic liver diseases in response to various liver-damaging factors. The main mechanisms of fibrotic or cirrhotic initiation and progression at the level of cellular and molecular events have been elucidated in the past two decades[1,2].

Improvement in erectile dysfunction after insulin-like growth factor-1 gene therapy in diabetic rats

Aim： To determine whether adenoviral gene transfer of insulin like growth factor-1 （IGF-1） to the penis of streptozotocin （STZ）-induced diabetic rats could improve erectile capacity. Methods： The STZ diabetic rats were transfected with AdCMV-βgal or AdCMV-IGF-1. These rats underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age-matched control rats 1 to 2 days after transfection. In control and transfected STZ diabetic rats, IGF-1 expression were examined by reverse transcription polymerase chain reaction （RT-PCR）, Western blot and histology. The penis β-galactosidase activity and localization of the STZ diabetic rats were also determined. Results： One to two days after transfection, the β-galactosidase was found in the smooth muscle cells of the diabetic rat penis transfected with AdCMV-βgal. One to 2 days after administration of AdCMV- IGF-1, the cavernosal pressure, as determined by the ratio of maximal intracavernous pressure-to-mean arterial pressure （ICP/MAP） and total intracavernous pressure （ICP）, was increased in response to cavernous nerve stimulation. Transgene expression was confirmed by RT-PCR, Western blot and histology. Conclusion： Gene transfer of IGF-1 significantly increased erectile function in the STZ diabetic rats. These results suggest that in vivo gene transfer of IGF- 1 might be a new therapeutic intervention for the treatment of erectile dysfunction （ED） in the STZ diabetic rats.

Adenoviral gene therapy in gastric cancer: A review

Gastric cancer is one of the most common malignancies worldwide. With current therapeutic approaches the prognosis of gastric cancer is very poor, as gastric cancer accounts for the second most common cause of death in cancer related deaths. Gastric cancer like almost all other cancers has a molecular genetic basis which relies on disruption in normal cellular regulatory mechanisms regarding cell growth, apoptosis and cell division. Thus novel therapeutic approaches such as gene therapy promise to become the alternative choice of treatment in gastric cancer. In gene therapy, suicide genes, tumor suppressor genes and anti-angiogenesis genes among many others are introduced to cancer cells via vectors. Some of the vectors widely used in gene therapy are Adenoviral vectors. This review provides an update of the new developments in adenoviral cancer gene therapy including strategies for inducing apoptosis, inhibiting metastasis and targeting the cancer cells.

p53 gene therapy in combination with transcatheter arterial chemoembolization for HCC:One-year follow-up

AIM:To evaluate the efficacy and safety of combination therapy with recombinant adenovirus p53 injection (rAdp53) and transcatheter hepatic arterial chemoembolization (TACE) for advanced hepatocellular carcinoma (HCC).METHODS:A total of 82 patients with advanced HCC treated only with TACE served as control group.Another 68 patients with HCC treated with TACE in combination with recombinant adenovirus-p53 injection served as p53 treatment group.Patients were followed up for 12 mo.Safety and therapeutic effects were evaluated according to the improvement in clinical symptoms,leukocyte count,Karnofsky and RECIST criteria.Survival rate was calculated with Kaplan-Meier method.RESULTS:The total effective rate was 58.3% for p53 treatment group,and 26.5% for control group (P < 0.05).The incidence of gastrointestinal symptoms was lower in p53 treatment group than in control group (P < 0.05).The 3-,6-and 12-mo survival rates were significantly higher for p53 treatment group than for control group (P < 0.01).The combination treatment was well tolerated with such adverse events as fever (51.5%,P=0.006) and pain of muscles and joints (13.2%,P=0.003),which were significantly higher than the chemotherapy.Except for these minor adverse effects,no severe vector-related complications were identified.With respect to the efficacy,patients in p53 treatment group had less gastrointerestinal symptoms (P=0.062),better improvement in tumor-related pain (P=0.003),less downgrade of leukocyte counts (P=0.003) and more upgrade of Karnofsky performance score (P=0.029) than those in control group.The total effective rate (CR + PR) for p53 treatment group and control group was 58.3% and 26.5%,respectively,with distributions of different effect in two groups (P=0.042).The survival rates were 89.71%,76.13%,and 43.30% for p53 treatment group,and 68.15%,36.98%,and 24.02% for control group,respectively,3,6 and 12 mo after treatment,suggesting that the survival rates are significantly higher for p53 treatment group than for control group (P=0.0002).CONCLUSION:The rAd-p53 gene therapy in combination with TACE is a safe and effective treatment modality for advanced HCC.

Therapy of cancer by cytokines mediated by gene therapy approach

Gene therapy offers a new approach for treatment of cancer. Transfer of genes encoding immunostimulatory cytokines has been used with remarkable success to eliminate cancer in animals. However, clinical trials in patients with this strategy had limited efficacy. Therefore, improvement ofgene transfer vector system is necessary. A hybrid viral vector, consisting of SFV replicon with either murine IL-12 or reporter LacZ gene, was constructed. This hybrid vector showed specificity and high level of expression in HCC both in vitro and in vivo. In a rat orthotropic liver tumor model, treatment of established tumors by the hybrid vector with raiL- 12 gene resulted in a strong anti-tumor activity without accompanying toxicity. Subsequently, a helper-dependent adenovirus vectors containing a mifepristone （RU486） inducible system was constructed for controlled and liver-specific expression of human interleukin 12 （hIL- 12） （HD-Ad/RUhIL- 12） and mouse IL-12 （mIL-12） （HD-Ad/RUmIL-12）. Data showed that high and sustained serum levels of hlL-12 could be attained by continuing administration of RU486 every 12 or 24 h. Repetitive induction ofhlL-12 could be obtained over, at least, a period of 48 weeks after a single injection of HD-Ad/RUhlL-12. Treatment of liver metastases with of HD-Ad/RUmIL- 12 plus RU846 resulted in complete tumor regression in all animals. Then, different cytokine genes were inserted into conditional replicative adenoviruses vectors （also called oncolytic adenovirus）. Replication ofadenovirus in tumor cells would kill tumor cells and release viruses, which infect surrounding tumor cells. The combination of cytopathic effect by oncolytic adenovirus and biological effect of transgene would exert strong antitumor activity. These new types of vectors may provide a potent and safe tool for cancer gene therapy.

Current gene therapy for stomach carcinoma

Gastric cancer is common in China [1-42],and its early diagnosis and treatment in advanced stage are difficult [31-50].In recent years ,gene study in cancer is a hotspot ,and great progress has been achieved [41-80] .Cancer gene therapy has shifted from the imagination into the laboratory and clinical trials.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Prevention of peritoneal adhesions:A promising role for gene therapy

Adhesions are the most frequent complication of abdominopelvic surgery,yet the extent of the problem,and its serious consequences,has not been adequately recognized.Adhesions evolved as a life-saving mecha-nism to limit the spread of intraperitoneal inflammatory conditions.Three different pathophysiological mechanisms can independently trigger adhesion formation.Mesothelial cell injury and loss during operations,tissue hypoxia and inflammation each promotes adhesion formation separately,and potentiate the effect of each other.Studies have repeatedly demonstrated that interruption of a single pathway does not completely prevent adhesion formation.This review summarizes the pathogenesis of adhesion formation and the results of single gene therapy interventions.It explores the prom-ising role of combinatorial gene therapy and vector modif ications for the prevention of adhesion formation in order to stimulate new ideas and encourage rapid advancements in this field.