Inhibition of graft arteriosclerosis development in rat aortas following heme oxygenase-1 gene transfer

Heme oxygenase 1 (HO-1) is an enzyme which degrades heme into tree end products: biliverdin, free iron and carbon monoxide. This enzyme has recently been shown to have anti-inflammatory and tissue protective effects. HO-1 expression is involved in organ protection in pathological situations, and immunosuppressive treatments resulting in indefinite graft survival without chronic rejection have been associated with HO-1 expression by cells of the vessel wall. The aim of this study was to analyze the effect of specific HO-1 overexpression. We used a recombinant adenovirus coding for human HO-1 cDNA in a rat aorta chronic rejection model, 30 days after transplantation. Control groups included rats non treated or treated with a non-coding adenovirus Addl324. We first demonstrated that AdHO-1 was efficiently expressed in endothelial cells in vitro, and in rat aortas ex vivo after adenovirus gene transfer. We found that intimal thickening in AdHO-1 treated aortas (10.8 +/- 3.8%, n=5) was significantly decreased compared to untreated (21.2 +/- 5.6%, n = 5) or Addl324-treated (21.1 +/- 1.2%, n = 4) aortas. Immunohistology showed that treatment with AdHO-1 resulted in a significant reduction in leukocyte infiltration and a decreasing number of VSMC in the intima, compared to Addl324-treated aortas. However, this effect of HO-1 on chronic rejection did not imply modifications on numbers of apoptotic cells in the graft or of alloantibody levels. We have demonstrated, for the first time, that specific HO-1 overexpression following gene transfer of HO-1 inhibited chronic rejection by reducing leukocyte and VSMC infiltration of the aorta intima.     

腺病毒介导的血红素氧合酶-1基因治疗对同种移植物慢性排斥反应损伤的保护作用

目的探讨腺病毒介导的血红素氧合酶-1（AdHO-1）基因治疗对同种移植物慢性排斥反应损伤的保护作用及其机制。方法选用血管移植和肾脏移植两种慢性排斥反应模型,对同种血管移植物和肾脏移植物进行体外AdHO-1基因转染,分析慢性排斥反应发生时移植物的结构和功能变化、目的基因和蛋白的表达以及免疫系统的相应反应。结果AdHO-1基因治疗缓解了慢性排斥反应对同种肾脏移植物的损伤,但弱于对血管移植物的保护效应;空载病毒加剧了同种肾脏移植物的损伤;AdHO-1基因治疗可减少慢性排斥反应发生时移植物内巨噬细胞和CD4^＋细胞的浸润。结论AdHO-1基因治疗可能通过保护移植物、下调免疫反应、诱导免疫偏移等作用减轻同种移植物的慢性排斥反应损伤。     

Induction of carbon monoxide in the donor reduces graft immunogenicity and chronic graft deterioration

Chronic allograft dysfunction remains the major obstacle for long-term successful transplantation. To date there is no effective treatment. Overexpression of protective genes has provided increased graft function and survival. This mechanism has been implicated in the process of graft accommodation. One of these genes that has been shown to mediate protective effects decodes the enzyme heme oxygenase-1 (HO-1), and an HO-1 downstream product, carbon monoxide (CO). Using an established model of kidney chronic allograft rejection in the rat, we investigated the impact of methylene chloride (MC), a CO donor, as a therapeutic tool to reduce chronic graft deterioration. We showed that donor and long-term recipient treatment with MC improved graft function and reduced histological signs of chronic rejection. Carbon monoxide may be a promising agent to improve graft quality and long-term graft function.     

Donor treatment with carbon monoxide can yield islet allograft survival and tolerance

Treatment of animals or certain cells with carbon monoxide (CO), a product of heme degradation by heme oxygenase-1 (HO-1), has potent anti-inflammatory and antiapoptotic effects that contribute to the survival of transplanted organs. We report here that inducing HO-1 in, or administering CO to, only the donor can be used in a therapeutic manner to sustain the survival of transplanted allogeneic islets. Similar treatments of only the islets or only the recipient are also salutary. Administering CO only to the donor frequently leads to long-term survival of those islets in untreated allogeneic recipients, which are then antigen-specifically tolerant. Several proinflammatory and proapoptotic genes that are strongly induced in islets after transplantation in the untreated situation were significantly suppressed after administering CO to the donor without further treatment. These included tumor necrosis factor-alpha, inducible nitric oxide synthase, monocyte chemoattractant protein-1, granzyme B, and Fas/Fas ligand, all of which contribute to the pathogenesis of the rejection of transplanted islets. This correlated with a lesser infiltration of recipient macrophages into the transplanted islets. Our present findings show that induction of HO-1 in, or administration of CO to, only the donor, islets, or the recipient or combinations of such treatments improve allogeneic islet survival.     

Systemic expression of heme oxygenase-1 ameliorates type 1 diabetes in NOD mice

Heme oxygenase-1 (HO-1) is an enzyme with potent immunoregulatory capacity. To evaluate the effect of HO-1 on autoimmune diabetes, female NOD mice at 9 weeks of age received a single intravenous injection of a recombinant adeno-associated virus bearing HO-1 gene (AAV-HO-1; 0.5 x 10(10)-2.5 x 10(10) viruses/mouse). In a dose-dependent manner, HO-1 transduction reduced destructive insulitis and the incidence of overt diabetes examined over a 15-week period. HO-1-mediated protection was associated with a lower type 1 T-helper cell (Th1)-mediated response. Adaptive transfer experiments in NOD.scid mice demonstrated that splenocytes isolated from AAV-HO-1-treated mice were less diabetogenic. Flow cytometry analysis revealed no significant difference in the percentages of CD4(+)CD25(+) regulatory T-cells between saline-treated and AAV-HO-1-treated groups. However, the CD11c(+) major histocompatibility complex II(+) dendritic cell population was much lower in the AAV-HO-1-treated group. A similar protective effect against diabetes was observed in NOD mice subjected to carbon monoxide (CO) gas (250 ppm CO for 2 h, twice per week). These data suggest that HO-1 slows the progression to overt diabetes in pre-diabetic NOD mice by downregulating the phenotypic maturity of dendritic cells and Th1 effector function. CO appears to mediate at least partly the beneficial effect of HO-1 in this disease setting.     

血红素加氧酶-1(HO-1)基因转染治疗急性胰腺炎的研究展望

血红素加氧酶-1(heme oxygenase-1,HO-1)通过分解血红素产生一氧化碳、胆绿素、胆红素及亚铁离子,可以减轻炎性介质反应,拮抗细胞氧化损伤和细胞应激从而发挥细胞保护作用.基因转染的方法能够维持目的基因的长时间表达,可以调节细胞因子的免疫反应.HO-1基因转染已在其他疾病的治疗研究中取得良好成果,本文针对...     

Protective effect of carbon monoxide for organ injury

Heme oxygenase (HO) catabolizes heme into three products: carbon monoxide (CO), biliverdin and free iron. HO-1, inducible form of HO, has been shown to be protective against various stress. CO at a low concentration has been shown to be protective in several disease models mimicking the action of HO-1. We suggest that CO, the byproduct of heme degradation, could be valuable therapeutic agents. We review the functional role of HO-1 and CO and its potential application to clinical settings.     

Peritransplant treatment with cobalt protoporphyrin attenuates chronic renal allograft rejection

Allogen-independent injury contributes to chronic rejection in renal allografts and heme oxygenase-1 (HO-1) has been shown to be protective in a number of settings. This study evaluated the effect of renal allograft recipient HO-1 up-regulation on chronic rejection in a rat model. Rat (F344 to Lewis) renal transplantation recipients were grouped: (i) cyclosporine (CsA) alone (0.75 mg/kg s.c. x 10 day; n = 5); (ii) CsA + low dose cobalt protoporphyrin (CoPP) an HO-1 inducer (0.5 mg/kg i.p. on days -5,0,5; n = 13) and (iii) CsA + high dose CoPP (5.0 mg/kg i.p. on days -5,0,5; n = 8). Renal function was assessed by serum creatinine levels on day 140. Histopathologic changes in allografts were graded. Morphometric analyses performed to objectively quantify the vascular changes and glomerulosclerosis. HO-1 expression quantified by Western blot and both HO-1 and endothelin (ET-1) localized using immunohistochemistry. Recipients treated with CsA + high dose CoPP had significantly decreased cortical scarring, vascular hyalinization and intimal thickness. They also had a significant, dose-dependent, reduction in luminal obliteration and glomerulosclerosis by morphometric analyses. This freedom from chronic rejection in recipients treated with CoPP translated into quiescent grafts at postoperative day 140 with immunostaining and Western blot demonstrating decreased level of HO-1 versus controls (P = 0.012). In summary, the peritransplant up-regulation of HO-1 in renal allograft recipients significantly attenuates chronic rejection in rat renal allografts by inhibiting transplant vasculopathy.     

Carbon Monoxide (CO) Protects Renal Tubular Epithelial Cells against Cold-Rewarm Apoptosis

Recent studies have suggested that carbon monoxide (CO) inhalation can reduce ischemia-reperfusion injury of kidneys. The purpose of the present study was to determine whether the direct application of CO using tricarbonylchloro (glycinato) ruthenium II (CORM3) would reduce cold-rewarm-associated apoptosis in renal tubular epithelial (RPTE) cells. RPTE cells were subjected to 48 hours of cold followed by 24 hours of rewarming with increasing concentrations (0–500 μM) of CORM3. CORM3 (100 μM) reduced apoptosis as determined by the TUNEL method from 21.6 ± 5.2 to 5.8 ± 1.1 % (untreated vs. treated, n?=?5; p < 0.001). We subsequently observed that the incubation of RPTE cells with CORM3 induced heme oxygenase (HO)-1 gene expression. As HO-1 itself can confer protection against cold rewarm injury, we investigated the role of HO-1 in the protective actions of CORM3 using siRNA oligonucleotides directed against HO-1. CORM3 treatment of RPTE cells caused a 4.9- fold increase in HO-1 gene expression as determined by real time PCR. Prior treatment of RPTE cells with siRNAs against HO-1 was able to completely abolish the CORM3 mediated induction of HO-1 mRNA and protein. The abolition of HO-induction with siRNAs did reduce CORM3-mediated protection against cold rewarm-induced apoptosis; however, CORM3 was able to significantly protect RPTE cells against cold-rewarm injury: apoptosis was 33.7 ± 0.9% vs. 15.4 ± 0.5% vs. 62.8 ± 1.5% vs. 23.5 ± 3.4 in control cold-rewarm vs. cold-rewarm + CORM3 (100 μM) vs. cold-rewarm + HO-1 siRNA vs. cold-rewarm + CORM3 (100 μM) + HO-1 siRNA (n?=?4). These results suggest that increased levels of CO alone can protect against cold-rewarm-induced apoptosis.     

Effect of a novel inducible nitric oxide synthase inhibitor in prevention of rat chronic aortic rejections

BACKGROUND: Cytotoxic nitric oxide (NO) is produced during ischemia-reperfusion injury and acute and chronic rejection in allografts by expression of inducible (i) NO synthase (NOS). Therefore, continuous inhibition of iNOS may prevent early graft dysfunction and immune injury (rejection) and consequently improve graft survival. FR260330 is a potent and selective inhibitor of iNOS activity that works by preventing iNOS monomers from dimerization. In this study, the authors evaluated the effect of FR260330 in prevention of chronic rejection in a model of rat aortic allografts. METHODS: Male Lewis (LEW, RT1l) rats received male ACI (RT1a) aorta allografts or LEW aorta isografts. Fourteen groups (n > or = 6) were involved in this study. FR260330, tacrolimus, or both were administered orally for 14 or 90 days, according to protocol. The degree of intimal proliferation of graft aorta was determined by a computerized image system. RESULTS: Both low and high doses of FR260330- or tacrolimus-treated grafts showed significantly decreased intima/(intima+media) ratios at day 90 compared with placebo controls. Combination therapy of low-dose FR260330 with low-dose tacrolimus produced a significant decrease of intima/(intima+media) ratios with intact endothelium compared with placebo controls. Anti-alpha-actin immunohistochemical staining demonstrated that one of the mechanisms of intimal proliferation is related to migration of vascular smooth muscle cells. CONCLUSIONS: A selective inhibitor of NOS, FR260330 plays a protective role in chronic aortic allograft rejection in the rat. Combination therapy of low-dose FR260330 with tacrolimus produces significant protection of immune injury and may serve to improve long-term graft survival and function.     

Egr-1 in vascular smooth muscle cell proliferation in response to allo-antigen

BACKGROUND: Early growth response factor-1 (Egr-1) plays an important role in regulating multiple factors involved in the progression of vascular lesions. This study examined our hypothesis that Egr-1 plays a critical role in the early stage of chronic cardiac allograft rejection and in the proliferation of the smooth muscle cell response to alloantigen. MATERIALS AND METHODS: Antisense Egr-1 oligodeoxynucleotide (ODN) was ex vivo gene transfected into the donor hearts from DBA/2 mice, followed by heterotopic allografting into B10.D2 recipients. The allografts were harvested on day 30. Egr-1 and its target molecules, such as platelet-derived growth factor (PDGF)-A, basic fibroblastic growth factor (bFGF), vascular cell adhesion molecule (VCAM)-1, transforming growth factor (TGF)-beta and nonmuscle myosin heavy chain B (SMemb), were identified immunohistochemically, and the percentage of the lumen occluded by the intima was calculated. For the cell proliferation assay, sensitized T cells were harvested from B10.D2 recipients as stimulator and then added to the SMCs, which were harvested from DBA/2 mouse aorta. Cellular proliferation was measured and Egr-1 and its target gene expression were examined by real-time RT-PCR. RESULTS: Egr-1 and its target genes were expressed in the thickened intima from untreated recipients. Egr-1 antisense ODN inhibited not only Egr-1 expression but also its target genes and significantly suppressed intimal thickening of coronary arteries. Egr-1 antisense ODN also significantly inhibited cell proliferation and expressions of Egr-1 and Egr-1 target genes in a mixed cell culture model. CONCLUSION: We conclude that Egr-1 plays an important role in the formation of the cardiac allograft vasculopathy responding to alloantigens.     

Heme oxygenase-1 system in organ transplantation

The heme oxygenase-1 (HO-1) system, the rate-limiting step in the conversion of heme, is among the most critical of cytoprotective mechanisms activated during cellular stress. The cytoprotection may result from the elimination of heme and the function of HO-1 downstream mediators, that is, biliverdin, carbon monoxide, and free iron. HO-1 overexpression exerts beneficial effects in a number of transplantation models, including antigen-independent ischemia/reperfusion injury, acute and chronic allograft rejection, and xenotransplantation. The HO-1 system is thought to exert four major functions: (1) antioxidant function; (2) maintenance of microcirculation; (3) modulatory function upon the cell cycle; and (4) anti-inflammatory function. The antioxidant function depends on heme degradation, oxygen consumption, biliverdin, and production of ferritin via iron accumulation. The production of carbon monoxide, which has vasodilation and antiplatelet aggregation properties, maintains tissue microcirculation and may be instrumental in antiapoptotic and cell arrest mechanisms. Heme catabolism and HO-1 overexpression exert profound direct and indirect inhibitory effects on the cascade of host inflammatory responses mediated by neutrophils, macrophages, and lymphocytes. These anti-inflammatory properties result in cytoprotection in a broad spectrum of graft injury experimental models, including ischemia/reperfusion, acute and chronic allograft, and xenotransplant rejection. Further, the multifaceted targets of HO-1-mediated cytoprotection may simultaneously benefit both local graft function and host systemic immune responses. Thus, the HO-1 system serves as a novel therapeutic concept in organ transplantation.     

Gene transfer of immunomodulatory peptides correlates with heme oxygenase-1 induction and enhanced allograft survival

BACKGROUND: Decapeptides derived from human HLA class I sequences have been shown to prolong allograft survival. The mechanism of action of these peptides has been uncertain, because they act in an MHC unrestricted manner. Recently, it was found that these peptides bind heme oxygenase 1 (HO-1). In the present study, we sought to determine whether local delivery of these peptides through gene transfer could extend allograft survival, and to explore the underlying mechanisms. METHODS: C57BL/6 neonatal hearts were transplanted to CBA/J recipients and the peptide, or plasmid DNA encoding the peptide, was injected directly into the allograft at the time of the transplant. RESULTS: Direct injection of 1 microg of the B2702 peptide into the allograft did not prolong survival (13.3+/-0.8 vs. 13.4+/-0.8 days for untreated controls), but injection of 400 microg of peptide did extend survival (22.0+/-0.6). Injection of plasmid DNA encoding the B2702 peptide was superior to peptide delivery, extending graft survival to 30.8+/-1.5 days. Similar results were obtained using another plasmid encoding the rationally designed peptide BC1 (28.5+/-1.7), whereas no significant prolongation was observed using a plasmid encoding the control peptide B2705 (16.5+/-1.0). To explore the hypothesis that these peptides exert their immunosuppressive effect by altering HO-1 activity, animals were treated with iron protoporphyrin, an inducer of HO-1 activity, or tin protoporphyrin, an inhibitor of HO-1. Treatment with iron protoporphyrin alone extended graft survival (24.5+/-1.6) and did not alter the benefit in survival seen with BC1 gene transfer (28.0+/-0.8). In contrast, treatment with tin protoporphyrin abolished the benefit of BC1 gene transfer (17.0+/-0.6). CONCLUSIONS: These results demonstrate that plasmid mediated gene transfer is an effective means for delivering immunosuppressive peptides to extend allograft survival. The experiments suggest that these peptides may act by increasing HO-1 activity and support a role for HO-1 in immune regulation and allograft survival.     

Carbon monoxide (CO) protects renal tubular epithelial cells against cold-rewarm apoptosis

Recent studies have suggested that carbon monoxide (CO) inhalation can reduce ischemia-reperfusion injury of kidneys. The purpose of the present study was to determine whether the direct application of CO using tricarbonylchloro (glycinato) ruthenium II (CORM3) would reduce cold-rewarm-associated apoptosis in renal tubular epithelial (RPTE) cells. RPTE cells were subjected to 48 hours of cold followed by 24 hours of rewarming with increasing concentrations (0-500 microM) of CORM3. CORM3 (100 microM) reduced apoptosis as determined by the TUNEL method from 21.6 +/- 5.2 to 5.8 +/- 1.1 % (untreated vs. treated, n = 5; p < 0.001). We subsequently observed that the incubation of RPTE cells with CORM3 induced heme oxygenase (HO)-1 gene expression. As HO-1 itself can confer protection against cold rewarm injury, we investigated the role of HO-1 in the protective actions of CORM3 using siRNA oligonucleotides directed against HO-1. CORM3 treatment of RPTE cells caused a 4.9- fold increase in HO-1 gene expression as determined by real time PCR. Prior treatment of RPTE cells with siRNAs against HO-1 was able to completely abolish the CORM3 mediated induction of HO-1 mRNA and protein. The abolition of HO-induction with siRNAs did reduce CORM3-mediated protection against cold rewarm-induced apoptosis; however, CORM3 was able to significantly protect RPTE cells against cold-rewarm injury: apoptosis was 33.7 +/- 0.9% vs. 15.4 +/- 0.5% vs. 62.8 +/- 1.5% vs. 23.5 +/- 3.4 in control cold-rewarm vs. cold-rewarm + CORM3 (100 microM) vs. cold-rewarm + HO-1 siRNA vs. cold-rewarm + CORM3 (100 microM) + HO-1 siRNA (n = 4). These results suggest that increased levels of CO alone can protect against cold-rewarm-induced apoptosis.     

Increased levels of exhaled carbon monoxide in bronchiectasis: a new marker of oxidative stress

BACKGROUND: Bronchiectasis is a chronic inflammatory lung disease associated with increased production of oxidants due mostly to neutrophilic inflammation. Induction of heme oxygenase (HO-1) by reactive oxygen species is a general cytoprotective mechanism against oxidative stress. HO-1 catabolises heme to bilirubin, free iron and carbon monoxide (CO). Exhaled CO measurements may therefore reflect an oxidative stress and be clinically useful in the detection and management of inflammatory lung disorders. METHODS: The levels of exhaled CO of 42 non-smoking patients with bronchiectasis treated or not treated with inhaled corticosteroids were compared with CO levels in 37 normal non-smoking subjects. RESULTS: Levels of exhaled CO were raised in patients with bronchiectasis, both those treated with inhaled corticosteroids (n = 27, median 5.5 ppm, 95% CI 5.16 to 7.76) and those not treated with inhaled corticosteroids (n = 15, median 6.0 ppm. 95% CI 4.74 to 11.8), compared with normal subjects (n = 37, median 3.0 ppm, 95% CI 2.79 to 3.81, p = 0.0024). There was no correlation between exhaled CO and HbCO levels (r = 0.42, p = 0.12) in normal subjects (n = 7), nor between the urine cotinine concentration and exhaled CO levels (r = 0.2, p = 0.12). CONCLUSIONS: Increased levels of exhaled CO may reflect induction of HO-1 and oxidative stress in bronchiectasis. Measurement of exhaled CO may be useful in the management of bronchiectasis and possibly other chronic inflammatory lung disorders.     

Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets

There is considerable interest in the viral modification of insulin-producing islets, including porcine islets, in the context of islet xenotransplantation to treat type 1 diabetes. Adenovirus (Adv) gene delivery offers the potential to modify pre-transplant islets for enhanced survival. Modifications include transfer of cytoprotective molecules to ensure islet survival immediately post-transplant, and molecules to dampen the immune system and prevent chronic islet graft rejection. In this study, we compared different promoters (three promiscuous and two tissue-specific promoters) for their efficiency in driving gene expression in neonatal pig islet tissue after Adv delivery. We also compared the efficiency of these promoters in adult islets from mouse and human pancreata. We observed that the promiscuous cytomegalovirus promoter was the most potent, eliciting high luciferase expression in neonatal pig islets, as well as in human and mouse islets. In contrast, the mammalian EF1-alpha promoter educed comparatively intermediate gene expression. The mouse major histocompatibility complex class I promoter H-2K(b) and the pancreatic-specific promoters insulin and human pdx-1 (area II) performed poorly in islets from all three species. This has important implications for the generation of modified neonatal pig islets for transplantation into humans.     

Study of gene delivery in a rabbit vein graft model. Improvement of the efficiency of gene transfer into vein grafts.

Gene therapy is a therapeutic strategy in treating cardiovascular disease. Vein graft failure, the major limitation on coronary artery bypass surgery, may be amenable to gene approaches. Some studies describe gene therapies using functioning genes to prevent vein graft stenosis. Gene transfer efficiency remains a major issue. In this rabbit vein graft model, we studied gene delivery using a replication-deficit recombinant adenovirus to improve gene transfer efficiency into vein grafts. The adenovirus vector that contains the E.coli lacZ gene encoding beta gal was used because this vector is widely used and thought to be effective. Gene transfer was detected by X-gal staining. We hypothesized that dimethylsulfoxide and hyaluronidase, both drug delivery enhancers, would improve efficiency and that, in transfer to adventitia, direct injection would be more effective than dwelling. We studied 3 gene delivery methods to intima and media (controls, using dimethylsulfoxide and using hyaluronidase before transfection) and 2 delivery methods to adventitia (direct injection and dwelling). We used 6 rabbits per delivery method. X-gal stained positive cell rates were counted using light microscopy. Our findings indicate that (1) dimethylsulfoxide increased the efficiency of transfection to media and intima, (2) hyaluronidase increased intimal transfection efficiency, (3) direct injection to adventitia was more effective than dwelling. These findings suggest that in vein grafting, our methods are feasible for improving gene transfer efficiency.     

Study of gene delivery in a rabbit vein graft model

Gene therapy is a therapeutic strategy in treating cardiovascular disease. Vein graft failure, the major limitation on coronary artery bypass surgery, may be amenable to gene approaches. Some studies describe gene therapies using functioning genes to prevent vein graft stenosis. Gene transfer efficiency remains a major issue. In this rabbit vein graft model, we studied gene delivery using a replication-deficit recombinant adenovirus to improve gene transfer efficiency into vein grafts. The adenovirus vector that contains the E.colilacZ gene encoding β gal was used because this vector is widely used and thought to be effective. Gene transfer was detected by X-gal staining. We hypothesized that dimethylsulfoxide and hyaluronidase, both drug delivery enhancers, would improve efficiency and that, in transfer to adventitia, direct injection would be more effective than dwelling. We studied 3 gene delivery methods to intima and media (controls, using dimethylsulfoxide and using hyaluronidase before transfection) and 2 delivery methods to adventitia (direct injection and dwelling). We used 6 rabbits per delivery method. X-gal stained positive cell rates were counted using light microscopy. Our findings indicate that (1) dimethylsulfoxide increased the efficiency of transfection to media and intima, (2) hyaluronidase increased intimai transfection efficiency, (3) direct injection to adventitia was more effective than dwelling. These findings suggest that in vein grafting, our methods are feasible for improving gene transfer efficiency.