高压尾静脉注射和位点特异性整合介导的人凝血因子Ⅸ基因治疗血友病B小鼠

目的 通过高压尾静脉注射将携带attB和人凝血因子Ⅸ(hFⅨ)的质粒与表达phiC31的质粒共同导入血友病B小鼠肝脏细胞,检测目的 质粒能否整合入小鼠基因组并持续表达.方法 ①构建表达hFⅨ并携带attB核心序列的真核表达载体attB-hFⅨ-pIRES2-EGFP,并在体外验证该载体能否表达目的 基因.②用高压尾静脉注射法将该载体与表达phiC31的质粒CMV-int共注入血友病B小鼠,attB-hFⅨ-pIRES2-EGFP单独注射为对照.③ELISA的方法检测hFⅨ在其体内的表达;用出血时间评价血友病小鼠出血症状是否改善;用巢式PCR检测attB-hFⅨ-pIRES2-EGFP是否整合到基因组的整合热点mpsL1(mouse pseudo-site from liver 1)位点.结果 经鉴定,attB-hFⅨ-pIRES2-EGFP载体构建成功,并在体外表达hFⅨ.高压尾静脉注入血友病B小鼠24 h后,hFⅨ血清水平达到最高值为(1533±239)ng/ml,此时小鼠的出血症状明显改善.但是不论是否与CMV-int共注射,此后hFⅨ水平迅速下降,在注射后10 d内降到本底水平.巢式PCR的结果证实,attB-hFⅨ-pIRES2-EGFP整合到小鼠肝脏基因组的mpsL1位点.结论 phiC31可以将34 bp的attB最短序列整合到小鼠基因组的整合热点mpsL1;由CMV启动hFⅨ的能够瞬间高表达并有效改善血友病小鼠的出血症状;但是外来DNA进入细胞后,无论是否整合到基因组均被迅速沉默,说明肺脏和肝脏对整合入基因组的CMV启动子表达调控的机制不尽相同,因此对用于基因治疗的裸DNA进行改进使其适合在靶器官表达是十分必要的.     

Long-term phenotypic correction in factor IX knockout mice by using ΦC31 integrase-mediated gene therapy

Hemophilia B, a hereditary bleeding disorder caused by a deficiency of coagulation factor IX (FIX), is an excellent candidate for gene therapy. However, to date, success in hemophilia gene therapy clinical trials has been limited due to failure to achieve or sustain therapeutic levels of factor expression. The ΦC31 integrase system efficiently integrates plasmid DNA carrying a transgene and an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, sustained transgene expression. A strategy utilizing plasmid DNA integrated with ΦC31 integrase may offer a facile and safe alternative for sustained human FIX (hFIX) expression. Hydrodynamic tail vein injection was used for delivery of plasmids encoding ΦC31 integrase and hFIX to the liver of FIX knockout mice. We demonstrated prolonged therapeutic levels of hFIX in this knockout mouse model of hemophilia B over a 6-month time course when ΦC31 integrase was used. Additionally, we observed sustained FIX activity in plasma and phenotypic correction of bleeding after tail clip in ΦC31-treated mice. In the livers that received integrase, we also demonstrated prolonged hFIX expression in hepatocytes by immunohistochemistry and documented sequence-specific genomic integration of the hFIX plasmid. These studies suggest the possibility that a similar approach in large animals and humans could lead to a simple and successful gene therapy for hemophilia.     

Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts.

Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.     

重组AAV2/hFⅨ病毒制备及其基因治疗血友病B的实验研究

目的　制备携带人凝血因子Ⅸ (hFⅨ )基因的重组AAV2病毒 (rAAV2 /hFⅨ ) ,并对用rAAV2 /hFⅨ肌肉注射治疗血友病B模型小鼠的疗效进行评价。方法　通过“一株载体细胞 /一株辅助病毒”的双因素包装策略制备出rAAV2 /hFⅨ ,体外转导BHK 2 1、C2C12细胞后 ,检测细胞培养上清中hFⅨ的表达量 ;肌肉直接注射血友病B模型小鼠后 ,检测其血浆中hFⅨ的抗原水平和凝血活性等指标。结果　转导 2 4h后在细胞上清中即可检测到hFⅨ ,连续检测 12 0h都有表达 ,BHK 2 1、C2C12细胞 2 4h最高表达量分别达到 (5 1.0± 6 .5 )ng/ 10 5细胞和 (6 8.0± 7.2 )ng/ 10 5细胞。rAAV2 /hFⅨ经肌肉直接注射后 ,高、中、低三个剂量组均能检测到小鼠体内高效表达hFⅨ ,在给药后第 3周达到高峰 ,小鼠血浆中hFⅨ的表达量与对照组比较差异有显著性 (P <0 .0 1) ,之后缓慢下降 ,到第 10周仍可检测到低水平hFⅨ表达 ;取第 3周小鼠血浆样品检测凝血功能 ,高、中、低剂量组FⅨ活性均得到明显改善 ,小鼠的割尾实验出血时间明显缩短 ,5min失血量也相应显著减少 ,其中高剂量组hFⅨ最高表达量达到 (387.0± 12 .5 )ng/ml血浆 ,FⅨ活性达到正常水平的 (30 .0± 5 .5 ) % ;给药后第 10周 ,除在注射点外 ,其它主要脏器均未检测到AAV载体DNA。结论     

Complete and sustained phenotypic correction of hemophilia B in mice following hepatic gene transfer of a high-expressing human factor IX plasmid

Summary.  Therapeutic correction of hemophilia B was achieved by rapid infusion of a large-volume solution containing a high-expressing human factor IX (hFIX) plasmid into the tail vein of hemophilia B mice. hFIX circulated at therapeutic levels (1–5 µg mL⁻¹) in all animals for more than 1 year as determined by both species-specific antigen assay and an activated partial thromboplastin time (APTT)-based clotting assay. There was acute, transient hepatic tissue damage by the infusion procedure and no significant inhibitory anti-hFIX antibodies developed. No bleeding episode was observed during or after treatment. Immunohistochemical studies indicated that the hFIX gene was exclusively expressed in hepatocytes, and that transduced cells had readily detectable hFIX protein at 4 h postinfusion, and stainable protein persisted for up to 1 year. Repeated infusions of hFIX plasmids boosted the hFIX expression to higher levels. These results demonstrate that hemophilia B can be treated by gene transfer of naked hFIX plasmids.     

PhiC31 integrase-mediated nonviral genetic correction of junctional epidermolysis bullosa

Patients afflicted with severe laminin 5-deficient junctional epidermolysis bullosa (JEB) often die in infancy with massive cutaneous blistering. Prior approaches to genetically correct this disorder have relied on stable integration of wild-type LAMB3 sequences, using retroviral vectors. To develop a nonviral approach to JEB gene therapy, we used the phiC31 integrase, which mediates unidirectional genomic integration of plasmids containing a specific attB site. An attB-containing laminin 5 beta3 expression plasmid was integrated into the genomes of primary keratinocytes from four unrelated, genetically characterized JEB patients. phiC31 integrase supported genomic integration into epidermal progenitor cells. Regeneration of human skin on immunedeficient mice, using these cells, produced human skin tissue with restored laminin 5 expression. Furthermore, corrected JEB tissue restored hemidesmosome formation and abolished histologic evidence of subepidermal blistering. These findings provide an approach to durable nonviral correction of JEB.     

Novel therapeutic approach for hemophilia using gene delivery of an engineered secreted activated Factor VII

Hemophilia is a bleeding disorder caused by mutations in the genes encoding coagulation Factor VIII (FVIII) or FIX. Current treatment is through intravenous infusion of the missing protein. The major complication of treatment is the development of neutralizing Ab's to the clotting factor. Infusion of recombinant activated human Factor VII (rhFVIIa), driving procoagulant reactions independently of human FVIII (hFVIII) or hFIX, has been successful in such patients and could in theory provide hemostasis in all hemophilia patients. However, its high cost and short half-life have limited its use. Here, we report a novel treatment strategy with a recombinant adeno-associated virus vector delivering a modified FVII transgene that can be intracellularly processed and secreted as activated FVII (FVIIa). We show long-term expression, as well as phenotypic correction of hemophilia B mice following gene transfer of the murine FVIIa homolog, with no evidence of thrombotic complications at these doses. These data hold promise for a potential treatment for hemophilia and other bleeding disorders.     

Molecular analysis of chromosomal rearrangements in mammalian cells after phiC31-mediated integration

Reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudoattP sites in the host chromosomes. In the present study we investigated whether cryptic phiC31 recognition sites in the host genome may result in chromosomal rearrangements. Of 69 independent integration events analyzed in human cells, 6 (8.7%) integrated into human chromosome 19 (19q13.31) and 10 (14.49%) integrated into human chromosome 12 (12q22). Most importantly, of all integration sites analyzed, 15% were found to contain an integrated transgene that was flanked by DNA sequences originating from two different chromosomes. To confirm chromosomal translocations we performed a polymerase chain reaction analysis of chromosomal DNA flanking the transgene and also performed limited studies to determine the genotype of single-cell clones. Although the mechanism responsible for chromosomal translocations needs to be further characterized, we speculate that cryptic phiC31 attachment sites flanking the transgene and cryptic phiC31 attachment sites in the host genome recombine with each other.     

Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage phiC31 integrase after nonviral DNA delivery

Peripheral vascular disease (PVD), characterized by insufficient blood supply to extremities, can be a devastating illness. Although many gene therapy strategies for PVD using vascular endothelial growth factor (VEGF) have resulted in increased blood vessel formation, the vessels are often impermanent and regress after therapy, probably because of the short-lived VEGF expression mediated by gene therapy vectors (14 days or less). phiC31 integrase is a recombinase originally isolated from a bacteriophage of Streptomyces. This integrase performs efficient chromosomal integration of plasmid DNA into mammalian genomes that results in long-term transgene expression. In this study, gene transfer was achieved by intramuscular injection of VEGF and integrase plasmid DNAs into the tibialis anterior muscle in the mouse hindlimb, followed by electroporation of the muscle with needle electrodes. We observed VEGF levels significantly above background 40 days after injection in animals that received phiC31 integrase and the VEGF plasmid. Site-specific integration of plasmid DNA in the chromosomes of muscle tissue was verified by polymerase chain reaction at a common integration site. These results suggest the possible utility of the phiC31 integrase system to treat ischemic disease.     

人源载体pHrnF9介导的凝血因子Ⅸ基因在肠上皮sw480细胞中的表达

本研究探索肠上皮细胞和人源载体pHrnFⅨ用于血友病B基因治疗的可能性。用含人凝血因子Ⅸ（human coagulation factorⅨ，hFⅨ）基因的人源载体质粒pHrnF9转染肠上皮细胞sw480，用RT-PCR检测mRNA的转录，荧光显微镜观察转染效率，ELISA和一期法检测蛋白表达及凝血活性。结果表明：转染后的细胞中有hFⅨmRNA的转录；荧光显微镜观察到48小时转染效率最高；ELISA法测得转染后24小时细胞上清中的hFⅨ蛋白量为（11．34±0．23）ng／（10^6cells·24h），第48小时hFⅨ蛋白量最高，达（29．34±1．00）ng／（10^6cells·24h），转染后72小时降为（12．45±0．15）ng）／（10^6cells·24h）。一期法结果显示转染pHrnF9的sw480细胞分泌的FⅨ有凝血活性，48小时达峰值（6．07±0、17）％／10^6cells，第72小时降至（1．81±0．06）％／10^6cells。结论：肠上皮细胞sw480转染pHrnF9质粒后可表达有凝血活性的hFⅨ，肠上皮细胞有望成为血友病B基因治疗的靶细胞。     

Induction of Immune Tolerance to FIX Following Muscular AAV Gene Transfer Is AAV-dose/FIX-level Dependent

Direct intramuscular injection (IM) of adeno-associated virus (AAV) has been proven a safe and potentially efficient procedure for gene therapy of many genetic diseases including hemophilia B. It is, however, contentious whether high antigen level induces tolerance or immunity to coagulation factor IX (FIX) following IM of AAV. We recently reported induction of FIX-specific immune tolerance by IM of AAV serotype one (AAV1) vector in mice. We hypothesize that the expression of high levels of FIX is critical to induction of FIX tolerance. In this study, we investigated the correlation among AAV dose, FIX expression, and tolerance induction. We observed that induction of immune tolerance or immunity to FIX was dependent on the dose of AAV1–human FIX (hFIX) given and the level of FIX antigen expressed in both normal and hemophilia mice. We then defined the minimum AAV1–hFIX dose and the lowest level of FIX needed for FIX tolerance. Different from hepatic AAV–hFIX gene transfer, we found that FIX tolerance induced by IM of AAV1 was not driven by regulatory T cells. These results provided further insight into the mechanism(s) of FIX tolerance, contributing to development of hemophilia gene therapy, and optimization of FIX tolerance induction protocols.     

Long-term expression of human coagulation factor VIII in a tolerant mouse model using the φC31 integrase system

We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the φC31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain. Various plasmids encoding φC31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Long-term expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type φC31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of φC31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and φC31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The φC31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia A.     

In vivo correction of murine hereditary tyrosinemia type I by phiC31 integrase-mediated gene delivery.

Phage phiC31 integrase is a site-specific recombinase that mediates efficient integration of circular extrachromosomal DNA into the host genome. Here, the integrase system was used to transfer the fumarylacetoacetate hydrolase (FAH) gene into the liver of mice affected with hereditary tyrosinemia type 1. Approximately 3.6% of transfected hepatocytes experienced an integration event. The absolute frequency of integration was 1/1374. A higher proportion of integrase-transfected FAH+ hepatocytes displayed abnormal morphology (bizarre nuclei, enlarged cells) on day 25 after gene transfer, compared to cells not receiving integrase. The increased frequency of these abnormal cells correlated with the amount of integrase plasmid administered, suggesting some form of integrase toxicity in Fah-/- livers. The abnormal hepatocyte appearance was transient and livers analyzed after longer selection (90 days) showed 60% repopulation with only normal healthy FAH+ hepatocytes. A total of seven different integration sites (accounting for >90% of integration) were identified. Serial transplantation of integrase-corrected hepatocytes to Fah-/- recipients was successful, suggesting long-term viability of corrected cells and persistent gene expression through many rounds of cell division. The stability of transgene expression, relatively high integration frequency, and significant site specificity that characterize the phiC31 integration system suggest that it may have utility in many gene therapy settings.     

Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer

BACKGROUND: The formation of inhibitory anti-factor IX (anti-FIX) antibodies is a major complication of FIX protein replacement-based treatment for hemophilia B. It is difficult to treat patients with anti-FIX antibodies. Gene therapy is emerging as a potentially effective treatment for hemophilia. Direct i.m. injection of adeno-associated virus (AAV) is a safe and efficient procedure for hemophilia B gene therapy. However, the development of anti-FIX antibodies following i.m. of AAV may impede its application to patients. OBJECTIVE: We aimed to investigate induction of immune tolerance to human FIX (hFIX) by i.m. of AAV1, further validating i.m. of AAV1 for hemophilia B gene therapy. METHODS AND RESULTS: Cohorts of hemostatically normal and hemophilia B mice with diverse genetic and MHC backgrounds received i.m. of AAV-hFIX. Human FIX antigen and anti-hFIX antibodies were examined. I.m. of 1 x 10(11) vector genomes (VG) of AAV2 elicits formation of anti-hFIX antibodies comparable to those by hFIX protein replacement. I.m. of 1 x 10(11) VG of AAV1 results in expression of therapeutic levels of hFIX (up to 950 ng mL(-1), mean = 772 ng mL(-1), SEM +/- 35.7) and hFIX-specific immune tolerance in C57BL/6 mice. CONCLUSIONS: A single i.m. of AAV1 can result in efficient expression of therapeutic levels of hFIX and induction of hFIX tolerance in hemostatically normal and hemophilic B mice. Our results substantiate the prospect of i.m. of AAV1 for hemophilia B gene therapy and FIX tolerance induction.     

Dystrophin expression in host muscle following transplantation of muscle precursor cells modified with the phiC31 integrase

Duchenne muscular dystrophy (DMD) is the most severe muscular dystrophy. It is caused by the absence of dystrophin in muscle fibers. The autologous transplantation of genetically corrected muscle precursor cells (MPCs) is a possible cure for DMD. A non-viral method of genetic modification was tested in this study. The co-transfection (nucleofection) of a phiC31 integrase and a transgene expressing plasmid in MPCs led to an increased stable expression in vitro. The stable expression of a small transgene (eGFP) in muscle fibers was initially demonstrated following the transplantation of the genetically modified cells. The stable expression of a truncated version of dystrophin as well as the full-length dystrophin fused with eGFP was then demonstrated in MPCs obtained from an mdx mice. The transplantation of these cells led not only to the expression of these fusion proteins in muscle fibers but also to the reconstitution of the dystrophin complex. Human MPCs were also genetically modified with a plasmid coding for the full-length human dystrophin gene fused with eGFP and transplanted in severe combined immuno deficient mice leading to the expression of eGFP dystrophin in muscle fibers. This work indicates that cell transplantation after correction of MPCs with phiC31 integrase is a possible approach to treat DMD.     

新型血友病B基因治疗的腺病毒-整合酶嵌合系统的构建及其体外表达鉴定

本研究构建一种既可高效感染靶细胞又能实现安全定点整合入宿主基因组的腺病毒嵌合载体。通过一系列DNA操作构建腺病毒-整合酶嵌合系统,该系统包含两个腺病毒载体:一个携带转基因表达框,表达框内有hFⅨ,红色荧光蛋白编码序列以及attB(phiC31识别位点),表达框两侧各有1个loxP(Cre识别位点);另一个腺病毒载体携带Cre和phiC31基因。同时构建只表达Cre和只表达phiC31的载体作为对照载体。在体外分别用脂质体转染293A细胞,用荧光显微镜观察荧光蛋白的表达,用RT-PCR鉴定各个基因的表达。结果表明,该腺病毒-整合酶嵌合系统构建成功。体外分别用脂质体转染293A细胞后,可见绿色荧光蛋白和红色荧光蛋白表达。RT-PCR鉴定表明,该系统能够成功表达各种目的蛋白。结论:成功构建了腺病毒-整合酶嵌合系统,为进一步研究其治疗作用提供良好基础。     

Balloon Catheter Delivery of Helper-dependent Adenoviral Vector Results in Sustained,Therapeutic hFIX Expression in Rhesus Macaques

Hemophilia B is an excellent candidate for gene therapy because low levels of factor IX (FIX) (≥1%) result in clinically significant improvement of the bleeding diathesis. Helper-dependent adenoviral (HDAd) vectors can mediate long-term transgene expression without chronic toxicity. To determine the potential for HDAd-mediated liver-directed hemophilia B gene therapy, we administered an HDAd expressing hFIX into rhesus macaques through a novel and minimally invasive balloon occlusion catheter-based method that permits preferential, high-efficiency hepatocyte transduction with low, subtoxic vector doses. Animals given 1 × 10¹² and 1 × 10¹¹ virus particle (vp)/kg achieved therapeutic hFIX levels for the entire observation period (up to 1,029 days). At 3 × 10¹⁰ and 1 × 10¹⁰ vp/kg, only subtherapeutic hFIX levels were achieved which were not sustained long-term. Balloon occlusion administration of HDAd was well tolerated with negligible toxicity. Five of six animals developed inhibitors to hFIX. These results provide important information in assessing the clinical utility of HDAd for hemophilia B gene therapy.     

Recent progress in gene therapy for hemophilia

Hemophilia A and B are X-linked monogenic disorders caused by deficiencies in coagulation factor VIII (FVIII) and factor IX (FIX), respectively. Current treatment for hemophilia involves intravenous infusion of clotting factor concentrates. However, this does not constitute a cure, and the development of gene-based therapies for hemophilia to achieve prolonged high level expression of clotting factors to correct the bleeding diathesis are warranted. Different types of viral and nonviral gene delivery systems and a wide range of different target cells, including hepatocytes, skeletal muscle cells, hematopoietic stem cells (HSCs), and endothelial cells, have been explored for hemophilia gene therapy. Adeno-associated virus (AAV)-based and lentiviral vectors are among the most promising vectors for hemophilia gene therapy. Stable correction of the bleeding phenotypes in hemophilia A and B was achieved in murine and canine models, and these promising preclinical studies prompted clinical trials in patients suffering from severe hemophilia. These studies recently resulted in the first demonstration that long-term expression of therapeutic FIX levels could be achieved in patients undergoing gene therapy. Despite this progress, there are still a number of hurdles that need to be overcome. In particular, the FIX levels obtained were insufficient to prevent bleeding induced by trauma or injury. Moreover, the gene-modified cells in these patients can become potential targets for immune destruction by effector T cells, specific for the AAV vector antigens. Consequently, more efficacious approaches are needed to achieve full hemostatic correction and to ultimately establish a cure for hemophilia A and B.     

A direct comparison of two nonviral gene therapy vectors for somatic integration: in vivo evaluation of the bacteriophage integrase phiC31 and the Sleeping Beauty transposase.

In this study we performed a head-to-head comparison of the integrase phiC31 derived from a Streptomyces phage and the Sleeping Beauty (SB) transposase, a member of the TC1/mariner superfamily of transposable elements. Mouse liver was cotransfused with a vector containing our most robust human coagulation factor IX expression cassette and the appropriate recombinase recognition site and either a phiC31- or a SB transposase-expressing vector. To analyze transgene persistence and to prove somatic integration in vivo we induced cell cycling of mouse hepatocytes and found that the transgene expression levels dropped by only 16 to 21% and 56 to 66% in mice that received phiC31 and SB, respectively. Notably, no difference in the toxicity profile was detected in mice treated with either recombinase. Moreover we observed that with the integrase-mediated gene transfer, transgene expression levels were dependent on the remaining noncoding vector sequences, which also integrate into the host genome. Further analyses of a hot spot of integration after phiC31-mediated integration revealed small chromosomal deletions at the target site and that the recombination process was not dependent on the orientation in which the phiC31 recognition site attached to the pseudo-recognition sites in the host genome. Coupled together with ongoing improvements in both systems this study suggests that both nonviral vector systems will have important roles in achieving stable gene transfer in vivo.