Hepatic Gene Therapy Research Articles

Hepatic gene therapy using lentiviral vectors: Has safety been established?

Hepatic gene therapy using lentiviral vectors: Has safety been established?

Hepatic lentiviral gene transfer is associated with clonal selection, but not with tumor formation in serially transplanted rodents

Lentiviral (LV) vectors are promising tools for long-term genetic correction of hereditary diseases. In hematopoietic stem cell gene therapies adverse events in patients due to vector integration-associated genotoxicity have been observed. Only a few studies have explored the potential risks of LV gene therapy targeting the liver. To analyze hepatic genotoxicity in vivo, we transferred the fumarylacetoacetate hydrolase (FAH) gene by LV vectors into FAH((-/-)) mice (n = 97) and performed serial hepatocyte transplantations (four generations). The integration profile (4,349 mapped insertions) of the LV vectors was assessed by ligation-mediated polymerase chain reaction and deep sequencing. We tested whether the polyclonality of vector insertions was maintained in serially transplanted mice, linked the integration sites to global hepatocyte gene expression, and investigated the effects of LV liver gene therapy on the survival of the animals. The lifespan of in vivo gene-corrected mice was increased compared to 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) control animals and unchanged in serially transplanted animals. The integration profile (4,349 mapped insertions) remained polyclonal through all mouse generations with only mild clonal expansion. Genes close to the integration sites of expanding clones may be associated with enhanced hepatocyte proliferation capacity. We did not find evidence for vector-induced tumors. LV hepatic gene therapy showed a favorable risk profile for stable and long-term therapeutic gene expression. Polyclonality of hepatocyte regeneration was maintained even in an environment of enforced proliferation.

114. A Novel Adeno-Associated Virus Serotype (AAV)-8 Capsid Mutant Improves Human Coagulation Factor IX Expression In Vivo

Recombinant AAV-8 vectors have shown significant promise for hepatic gene therapy of hemophilia B. However, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. We hypothesized that AAV8 during its intracellular trafficking, are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery and modification of specific serine/threonine kinase or ubiquitination targets on AAV8 capsid (Fig.1A) may improve its transduction efficiency. To test this, point mutations at specific serine (S)/threonine (T) > alanine (A) or lysine (K)>arginine (R) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant(S276A, S501A, S671A, T251A and K137R) capsids were evaluated for their liver transduction efficiency at a dose of 5 X 1010 vgs/ animal in C57BL/6 mice in vivo. The best performing mutant was found to be the K137R vector in terms of either the gene expression (46-fold) or the vector copy numbers in the hepatocytes (22-fold) compared to WT-AAV8 (Fig.1B). The K137R-AAV8 vector that showed significantly decreased ubiquitination of the viral capsid had reduced activation of markers of innate immune response [IL-6, IL-12, tumor necrosis factor α, Kupffer cells and TLR-9]. In addition, animals injected with the K137R mutant also demonstrated decreased (2-fold) levels of cross-neutralizing antibodies when compared to animals that received the WT-AAV8 vector. To study further the utility of the novel AAV8-K137R mutant in a therapeutic setting, we delivered human coagulation factor IX (h.FIX) under the control of liver specific promoters (LP1 or hAAT) at two different doses (2.5x10^10 and 1x10^11 vgs per mouse) in 8-12 weeks old male C57BL/6 mice. As can be seen in Fig.1C/D, the circulating levels of h.FIX were higher in all the K137R-AAV8 treated groups as compared to the WT-AAV8 treated groups either at 2 weeks (62% vs 37% for hAAT constructs and 47% vs 21% for LP1 constructs) or 4 weeks (78% vs 56% for hAAT constructs and 64% vs 30% for LP1 constructs) post hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel vector for potential gene therapy of hemophilia B.

Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiencyin Vitroandin Vivo

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T→alanine [A] or 9 K→arginine [R] single mutant or 2 double K→R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T→A and 7 K→R vectors were significantly higher (~63-90%) than the AAV2-WT vectors (~30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated ~8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.

Human thyroxine binding globulin (TBG) promoter directs efficient and sustaining transgene expression in liver-specific pattern

The liver performs a vital role in metabolic process, which makes it an attractive target organ for gene therapy. To improve the effects of gene therapy in disorders caused by metabolic disturbance, we quantitatively evaluated six promoters, CMV, EF1α, PGK, apoE, thyroxine binding globulin (TBG), and cytochrome P450 2E1 (CYP2E1) by measuring the expression of α1-antitrypsin, which is controlled by these promoters and introduced via a lentivirus-mediated delivery system in the liver. The results showed that the TBG promoter presents as highly active though in general it is slightly lower than the ubiquitous CMV and EF1α. The expression of exogenous genes driven by the TBG promoter demonstrates to be much higher than by PGK, apoE, and CYP2E1 promoters, and the fragment of −435bp to −26bp from transcription start site (TSS) in the TBG promoter region is identified as the optimum region to direct transgene expression at a higher level. In addition, we further confirmed that the TBG promoter confers transgene persistent and specific expression within the liver up to several months after integration. The data suggests that the TBG promoter is a valuable tool and will greatly facilitate the optimization of vector design in hepatic gene therapy.

Study of the construction of an adenoviral vector for hepatic tumor gene therapy application

Study of the construction of an adenoviral vector for hepatic tumor gene therapy application

Effects of hepatic insulin gene therapy on enteric neuropathy in STZ-diabetic mice

To evaluate the effect of hepatic insulin gene therapy on diabetic enteric neuropathy. Mice were randomly allocated into 3 groups: a normal control group, a diabetic group, and a diabetic gene therapy group. Diabetes were induced by penial vein injection of streptozocin (STZ). The gene therapy group received hepatic insulin gene therapy while the other 2 groups only received an empty virus expressing green fluorescent protein. Random blood glucose, body weight growth, gastric emptying, total bowel length, absolute and relative bowel transit, electric field stimulation of colon smooth muscle, colon nuclei staining and counting were measured. We successully established a mouse model of diabetic enteric neuropathy which manifests as: 8 weeks of continuous hyperglycemia,increased total bowel length, decreased relative bowel transit, impaired colon smooth muscle relaxation and loss of inhibitory neurons in colon. Through gene therapy, the above indexes were normalized or ameliorated, suggesting hepatic insulin gene therapy is capable of preventing diabetic enteric neuropathy. Hepatic insulin gene therapy can prevent STZ induced diabetic enteric neuropathy.

AAV-mediated gene therapy for liver diseases: the prime candidate for clinical application?

Introduction: Diseases of the liver represent a major health problem. Often treatments are ineffective, prompting the need for new therapeutic strategies. From extensive preclinical studies, gene therapy in particular mediated by adeno-associated virus (AAV)-derived vectors, has now emerged as the prime candidate for clinical application. AAV-mediated gene therapy for inherited liver diseases has now become a clinical reality, in particular for the treatment of hemophilia B.Areas covered: This review provides a summary of current literature on AAV-mediated gene therapies for both inherited and acquired liver diseases and outlines different strategies to overcome current clinical limitations. The unique properties of AAV over other viral vectors are highlighted as well as the current challenges which are faced for wide-ranging clinical application.Expert opinion: Despite the extensive positive results from animal models, successful application in clinical settings is hampered by immunological barriers. However, immune suppression and other strategies can be employed to overcome these limitations. Given some of their unique advantages, AAV vectors are currently the most obvious candidate for hepatic gene therapy applications, however, serotype-related issues of immune reactivity still represent a formidable barrier for clinical success.

539. The Role of TGF-β and IL-10 in the Development of Immune Tolerance Following Hepatic Gene Therapy

539. The Role of TGF-β and IL-10 in the Development of Immune Tolerance Following Hepatic Gene Therapy

Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice

Insulin deficiency in type 1 diabetes leads to disruptions in glucose, lipid, and ketone metabolism with resultant hyperglycemia, hyperlipidemia, and ketonemia. Exogenous insulin and hepatic insulin gene therapy cannot mimic the robust glucose-stimulated insulin secretion (GSIS) from native pancreatic islets. Gene therapy of streptozotocin-diabetic mice with neurogenin 3 (Ngn3) and betacellulin (Btc) leads to the induction of periportal oval cell-derived neo-islets that exhibit GSIS. We hence hypothesized that this gene therapy regimen may lead to a complete correction of the glucose and lipid metabolic abnormalities associated with insulin deficiency; we further hypothesized that the neo-islets formed in response to Ngn3-Btc gene delivery may display an ultrastructure and transcription profile similar to that of pancreatic islets. We injected streptozotocin-diabetic mice with helper-dependent adenoviral vectors carrying Ngn3 and Btc, which restored GSIS and reversed hyperglycemia in these animals. The treatment also normalized hepatic glucose secretion and reversed ketonemia. Furthermore, it restored hepatic glycogen content and reinstated hepatic lipogenesis-related gene transcripts back to nondiabetic levels. By transmission electron microscopy, the neo-islets displayed electron-dense granules that were similar in appearance to those in pancreatic islets. Finally, using RNA obtained by laser capture microdissection of the periportal neo-islets and normal pancreatic islets, we found that the neo-islets and pancreatic islets exhibited a very similar transcription profile on microarray-based transcriptome analysis. Taken together, this indicates that Ngn3-Btc gene therapy corrects the underlying dysregulated glucose and lipid metabolism in insulin-deficient diabetic mice by inducing neo-islets in the liver that are similar to pancreatic islets in structure and gene expression profile.

Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice

Insulin deficiency in type 1 diabetes leads to disruptions in glucose, lipid, and ketone metabolism with resultant hyperglycemia, hyperlipidemia, and ketonemia. Exogenous insulin and hepatic insulin gene therapy cannot mimic the robust glucose-stimulated insulin secretion (GSIS) from native pancreatic islets. Gene therapy of streptozotocin-diabetic mice with neurogenin 3 (Ngn3) and betacellulin (Btc) leads to the induction of periportal oval cell-derived neo-islets that exhibit GSIS. We hence hypothesized that this gene therapy regimen may lead to a complete correction of the glucose and lipid metabolic abnormalities associated with insulin deficiency; we further hypothesized that the neo-islets formed in response to Ngn3-Btc gene delivery may display an ultrastructure and transcription profile similar to that of pancreatic islets. We injected streptozotocin-diabetic mice with helper-dependent adenoviral vectors carrying Ngn3 and Btc, which restored GSIS and reversed hyperglycemia in these animals. The treatment also normalized hepatic glucose secretion and reversed ketonemia. Furthermore, it restored hepatic glycogen content and reinstated hepatic lipogenesis-related gene transcripts back to nondiabetic levels. By transmission electron microscopy, the neo-islets displayed electron-dense granules that were similar in appearance to those in pancreatic islets. Finally, using RNA obtained by laser capture microdissection of the periportal neo-islets and normal pancreatic islets, we found that the neo-islets and pancreatic islets exhibited a very similar transcription profile on microarray-based transcriptome analysis. Taken together, this indicates that Ngn3-Btc gene therapy corrects the underlying dysregulated glucose and lipid metabolism in insulin-deficient diabetic mice by inducing neo-islets in the liver that are similar to pancreatic islets in structure and gene expression profile.

Hepatic insulin gene therapy diminishes liver glycogen despite insulin responsive transcriptional effects in diabetic CD‐1 mice

Hepatic insulin gene therapy (HIGT) produces near-normal glycemia in diabetic rats. Hepatic insulin production is expected to stimulate glycogen storage. However, the effect of HIGT on hepatic glycogen metabolism in vivo is unknown. After administration of an adenoviral vector capable of inducing glucose responsive insulin production from hepatocytes, we evaluated circulating hormones, cytokines, hepatic gene expression and hepatic glycogen content in diabetic CD-1 mice receiving intravenous streptozotocin. Nondiabetic mice and diabetic mice treated with empty adenovirus served as controls. Peripheral concentrations of human insulin in HIGT mice were less than concentrations of mouse insulin among controls. However, expression of insulin responsive genes in HIGT livers indicated a significant intra-hepatic insulin effect, with expression changes reflecting appropriate responses to fed-fasting transitions. Transcription factors (hepatocyte nuclear factor-4alpha and peroxisome proliferator-activated receptor gamma co-activator-1alpha), as well as target genes (phospho-enol-pyruvate carboxykinase, glucose-6-phosphatase and glucokinase) exhibited insulin responsive expression. Despite producing near normal glycemia, HIGT diminished hepatic glycogen content in both fasted and fed mice. Serum cytokine responses revealed both vector-related (monocyte chemoatractant protein-1, interleukin-6) and transgene specific (resistin, tumor necrosis factor alpha) effects. HIGT produces low circulating concentrations of insulin, but produces significant intra-hepatic effects on gene expression. Despite controlling hyperglycemia, HIGT exerts unexpected insulin effects on hepatic carbohydrate metabolism. Although the precise mechanisms remain to be determined, they may relate to vector-induced cytokine effects.

Hepatic Insulin Gene Therapy Normalizes Diurnal Fluctuation of Oxidative Metabolism in Diabetic BB/Wor Rats

Previous studies of hepatic insulin gene therapy (HIGT) focused on glycemic effects of insulin produced from hepatocytes. In this study, we extend the observations of glycemic control with metabolically regulated HIGT to include systemic responses and whole-body metabolism. An insulin transgene was administered with an adenoviral vector [Ad/(GlRE)(3)BP1-2xfur] to livers of BB/Wor rats made diabetic with polyinosinic polycytidilic acid (poly-I:C) (HIGT group), and results compared with nondiabetic controls (non-DM), and diabetic rats receiving different doses of continuous-release insulin implants (DM-low BG and DM-high BG). Blood glucose and growth normalized in HIGT, with lower systemic insulin levels, elevated glucagon, and increased heat production compared with non-DM. Minimal regulation of systemic insulin levels were observed with HIGT, yet the animals maintained normal switching from carbohydrate to lipid metabolism determined by respiratory quotients (RQs), and tolerated 24-hour fasts without severe hypoglycemia. HIGT did not restore serum lipids as we observed increased triglycerides (TGs) and increased free fatty acids, but reduced weight of visceral fat pads despite normal total body fat content and retroperitoneal fat depots. HIGT favorably affects blood glucose, normalizes metabolic switching in diabetic rats, and reduces intra-abdominal fat deposition.

Gene Gun Bombardment with DNA-Coated Gold Particles Is a Potential Alternative to Hydrodynamics-Based Transfection for Delivering Genes into Superficial Hepatocytes

Although in vivo nonviral gene delivery to the liver is critical for hepatic gene therapy, there are a number of technical obstacles. Enhanced green fluorescent protein (EGFP)-encoding DNA was coated onto gold particles (gold-DNA), dissolved in phosphate-buffered saline (pure DNA), and prepared as a polymer adjuvant (jetPEI)-galactosidase solution (polymer-DNA). Murine liver transfection was attempted by nonviral approaches, which included hydrodynamics-based transfection (HBT) of pure DNA, transport and transhepatic injection of polymer-DNA, and gene gun bombardment with pure DNA, gold-DNA, and polymer-DNA. Only HBT and gene gun bombardment yielded significant numbers of EGFP(+) hepatocytes. With the exception of the edge of the liver, HBT had a whole-liver transfection rate of 20% under optimized conditions. HBT resulted in marked hepatic infarctions, most prominently at the edge of the liver. For gene gun bombardment, the transfection rate was pressure dependent and limited to 15% for gold-DNA. Triple or quadruple bombardment at 30 psi resulted in a transfection rate comparable to that of a single bombardment at higher pressure, but was associated with minimal scattered hepatic necrosis. The EGFP(+) hepatocytes were located mainly in the superficial layers. We conclude that both HBT and gene gun bombardment yielded efficient murine hepatocyte transfection in vivo. Severe hepatic infarction impedes foreign gene expression in the superficial hepatocytes after HBT. Repeated bombardment with gold-DNA, using an accelerated particle gene gun at 30 psi, is a potential alternative to HBT for delivering genes to superficial hepatocytes in vivo, although gold-related hepatic necrosis is a persistent problem.

Glucose- and Metabolically Regulated Hepatic Insulin Gene Therapy for Diabetes

The purpose of this study was to examine glucose- and metabolically modulation of insulin secretion by rAAV-mediated gene delivery in vitro and in vivo. A recombinant adeno-associated virus vector (rAAV) containing a furin-mutated human insulin gene, driven by the rat insulin I promoter, was used in this study. Glucose-responsive secretion of human insulin was determined by treating rAAV-transduced Huh7 human hepatoma cells with varying concentrations of glucose, with or without insulin secretagogues. Glucose- and metabolically modulated secretion of human insulin in the streptozotocin (STZ)-induced diabetic mice was assessed by intrahepatic administration of rAAV-polyethylenimine (PEI) complexes, followed by intraperitoneal glucose tolerance test (IPGTT), with or without theophylline. Glucose- and metabolically controlled human insulin secretion was obtained in the rAAV-transduced Huh7 cells. Treatment of STZ-induced diabetic animals with rAAV-polyethylenimine (rAAV-PEI) complexes resulted in production of human insulin and amelioration of hyperglycemia. Co-administration of glucose and theophylline in these animals augmented the secretion of human insulin, demonstrating metabolic modulation of insulin secretion in vivo. Immunohistochemical examination of the liver sections of rAAV-treated mice confirmed the production of human insulin. Glucose- and metabolically controlled hepatic insulin gene therapy was obtained both in vitro and in vivo.

Calcium Phosphate—DNA Nanocomposites: Morphological Studies and Their Bile Duct Infusion for Liver‐Directed Gene Therapy

In this study, liver‐directed gene transfer in rats with calcium phosphate (CaP) nanoparticles and the effect of the route of administration and surgical manipulations on transfection efficiency is reported. Formulations of CaP nanoparticles entrapping plasmid DNA (pDNA) were prepared by the reverse micellar method using two different surfactants. Transmission electron microscopy, scanning electron microscopy and dynamic light scattering were used to characterize the CaP–DNA nanocomposites. The morphological characteristics of the formulations showed a strong dependency on temperature. Gel electrophoresis experiments indicated that there was no degradation of the encapsulated pDNA, and in vitro cell transfection in HEK‐293 and primary hepatocytes from rats as well as in vivo intraductal delivery experiments suggested that CaP nanoparticles led to significant and prolonged transgene expression. Therefore, our methodology gives a stable and viable formulation for hepatic gene therapy. Low‐DNA dosage entrapped in CaP nanoparticles makes it an effective gene delivery system for clinical applications.

Lentiviral vector hepatic insulin gene therapy resulted in the long-term attenuation of type 1 diabetes mellitus in an animal model,

Lentiviral vector hepatic insulin gene therapy resulted in the long-term attenuation of type 1 diabetes mellitus in an animal model,

Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy

Aims/hypothesisType 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral vectors are promising tools for liver-directed gene therapy. However, to date, transduction rates in vivo remain low in hepatocytes, without the induction of cell cycling. We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats.Materials and methodsTo improve transduction efficiency in vitro, we optimised hepatocyte isolation and maintenance protocols and, using an improved surgical delivery method, delivered furin-cleavable insulin alone or empty vector to the livers of streptozotocin-induced diabetic rats by means of a lentiviral vector. Rats were monitored for changes in body weight and blood glucose, and intravenous glucose tolerance tests were performed. Expression of insulin was determined by RT-PCR, immunohistochemistry and electron microscopy.ResultsWe achieved long-term transgene expression in quiescent hepatocytes in vitro (87 ± 1.2% transduction efficiency), with up to 60 ± 3.2% transduction in vivo. We normalised blood glucose for 500 days—a significantly longer period than previously reported—making this the first successful study using a lentiviral vector. This procedure resulted in the expression of genes encoding several beta cell transcription factors, some pancreatic endocrine transdifferentiation, hepatic insulin storage in granules, and restoration of glucose tolerance. Liver function tests remained normal. Importantly, pancreatic exocrine transdifferentiation did not occur.Conclusions/interpretationOur data suggest that this regimen may ultimately be employed for the treatment of type 1 diabetes.

Engineered Enteroendocrine Cells Secrete Insulin in Response to Glucose and Reverse Hyperglycemia in Diabetic Mice

Type 1 diabetes is a metabolic disorder caused by loss of insulin-producing pancreatic beta-cells. Expression of insulin in non-beta-cells to create beta-cell surrogates has been tried to treat type 1 diabetes. Enteroendocrine K cells have characteristics similar to pancreatic beta-cells, such as a glucose-sensing system and insulin-processing proteases. In this study, we genetically engineered an enteroendocrine cell line (STC-1) to express insulin under the control of the glucose-dependent insulinotropic polypeptide promoter. We screened clones and chose one, Gi-INS-7, based on its high production of insulin. Gi-INS-7 cells expressed glucose transporter 2 (GLUT2) and glucokinase (GK) and secreted insulin in response to elevated glucose levels in vitro. To determine whether Gi-INS-7 cells can control blood glucose levels in diabetic mice, we transplanted these cells under the kidney capsule of streptozotocin (STZ)-induced diabetic mice and found that blood glucose levels became normal within 2 weeks of transplantation. In addition, glucose tolerance tests in mice that became normoglycemic after transplantation with Gi-INS-7 cells showed that exogenous glucose was cleared appropriately. These results suggest that engineered K cells may be promising surrogate beta-cells for possible therapeutic use for the treatment of type 1 diabetes.

175: A New Paradigm for Tissue Regeneration: Preparative Irradiation for Cell-Based Therapies as an Alternative to Organ Transplantation

Orthotopic liver transplantation (OLT) is the only curative therapy for patients with terminal liver diseases. A significant number of patients fail to get OLT due to shortage of donor livers. Hepatocyte transplantation (HT) is being explored as a therapeutic alternative to OLT for the treatment of end-stage liver diseases. However, the number of cells that can be safely transplanted into the liver and the inability of the transplanted hepatocytes to proliferate in the host liver currently limit the clinical application of this procedure. We hypothesized that a preparative regimen of hepatic irradiation (HIR), in combination with hepatic mitotic stimuli, would suppress host hepatocellular proliferation and induce post-mitotic death, thus ”making room“ for transplanted, nonirradiated hepatocytes to proliferate preferentially and repopulate the irradiated host liver. C57Bl/6 mice received HIR (0-50 Gy) to the anterior liver lobes after exposing the liver by laparotomy and shielding the posterior liver lobes and other abdominal organs, followed by an injection of a recombinant adenoviral vector expressing human hepatocyte growth factor (Ad-HGF, 5-10 x 1010 particles). One day after HIR, the mice received intrasplenic HT with 0.8-1×106 primary hepatocytes, freshly isolated from congenic, beta-galactosidase-expressing transgenic (ROSA)26 C57Bl/6 mice. Control mice received Ad-HGF alone, or HIR + Ad-LacZ with HT. Animals were sacrificed at 7 days, 1, 2, 4 and 6 months after HT and the liver sections were examined for beta-galactosidase-expressing donor cells after histochemical and hematoxylin-eosin staining. Compared to unirradiated controls, HIR augmented the engraftment of transplanted hepatocytes, with an increase in the number of integrated donor hepatocytes into liver cords by 7 days (p<0.01). There was progressive repopulation of the irradiated anterior liver lobes by transplanted hepatocytes with near-total (80-95%) replacement of the host hepatocytes by the donor cells within 6 months. In contrast, the unirradiated posterior and caudate liver lobes exhibited occasional presence of beta-galactosidase-expressing donor hepatocytes, indicating engraftment without repopulation. H&E staining demonstrated normal liver architecture without any evidence of hepatic radiation injury. Liver function tests of the transplanted animals were normal. Preparative irradiation is routinely used for bone marrow transplantation, but we are the first to use HIR to facilitate the engraftment and host liver repopulation by donor cells after HT in rodent models. HIR significantly enhanced the integration of transplanted cells in the host liver. Furthermore, partial liver irradiation enabled selective repopulation of a liver lobe by the donor cells, thereby increasing the safety of our regimen. Clinically, preparative HIR could be safely administered using radiosurgery or 3-D conformal RT and combined with systemic administration of hepatic growth factors to facilitate HT for 1) the treatment of inherited liver diseases, 2) liver failure, 3) ex vivo hepatic gene therapy, 4) rescuing patients with liver cancer, following chemo-radiation therapy, and 5) expanding human hepatocytes in animal liver for generating animal models for human-specific infections.