Safe Procedure for Efficient Hydrodynamic Gene Transfer to Isolated Porcine Liver in Transplantation.

Although calcineurin inhibitors are very effective as immunosuppressants in organ transplantation, complete graft acceptance remains as a challenge. Transfer of genes with immunosuppressant functions could contribute to improving the clinical evolution of transplantation. In this sense, hydrodynamic injection has proven very efficacious for liver gene transfer. In the present work, the hIL-10 gene was hydrofected 'ex vivo' to pig livers during the bench surgery stage, to circumvent the cardiovascular limitations of the procedure, in a model of porcine orthotopic transplantation with a 10-day follow-up. We used IL-10 because human and porcine proteins can be differentially quantified and for its immunomodulatory pleiotropic functions. Safety (biochemical parameters and histology), expression efficacy (RNA transcription and blood protein expression), and acute inflammatory response (cytokines panel) of the procedure were evaluated. The procedure proved safe as no change in biochemical parameters was observed in treated animals, and human IL-10 was efficaciously expressed, with stationary plasma protein levels over 20 pg/mL during the follow-up. Most studied cytokines showed increments (interferon-α, IFN-α; interleukin-1ß, IL-1ß; tumor necrosis factor α, TNFα; interleukin-6, IL-6; interleukin-8, IL-8; interleukin-4, IL-4; and transforming growth factor-ß, TGF-ß) in treated animals, without deleterious effects on tissue. Collectively, the results support the potential clinical interest in this gene therapy model that would require further longer-term dose-response studies to be confirmed.

Personalized Medicine in Infant Population with Cancer: Pharmacogenetic Pilot Study of Polymorphisms Related to Toxicity and Response to Chemotherapy.

BACKGROUND: Pharmacogenetics is a personalized medicine tool that aims to optimize treatments by adapting them to each individual's genetics, maximizing their efficacy while minimizing their toxicity. Infants with cancer are especially vulnerable, and their co-morbidities have vital repercussions. The study of their pharmacogenetics is new in this clinical field. METHODS: A unicentric, ambispective study of a cohort of infants receiving chemotherapy (from January 2007 to August 2019). The genotypes of 64 patients under 18 months of age were correlated with severe drug toxicities and survival. A pharmacogenetics panel was configured based on PharmGKB, drug labels, and international experts' consortiums. RESULTS: Associations between SNPs and hematological toxicity were found. Most meaningful were: MTHFR rs1801131 GT increasing the anemia risk (OR 1.73); rs1517114 GC, XPC rs2228001 GT, increasing neutropenia risk (OR 1.50 and 4.63); ABCB1 rs1045642 AG, TNFRSF11B rs2073618 GG, CYP2B6 rs4802101 TC and SOD2 rs4880 GG increasing thrombocytopenia risk (OR 1.70, 1.77, 1.70, 1.73, respectively). Regarding survival, MTHFR rs1801133 GG, TNFRSF11B rs2073618 GG, XPC rs2228001 GT, CYP3A4 rs2740574 CT, CDA rs3215400 del.del, and SLC01B1 rs4149015 GA were associated with lower overall survival probabilities (HR 3.12, 1.84, 1.68, 2.92, 1.90, and 3.96, respectively). Lastly, for event-free survival, SLC19A1 rs1051266 TT and CDA rs3215400 del.del increased the relapse probability (HR 1.61 and 2.19, respectively). CONCLUSIONS: This pharmacogenetic study is a pioneer in dealing with infants under 18 months of age. Further studies are needed to confirm the utility of the findings in this work to be used as predictive genetic biomarkers of toxicity and therapeutic efficacy in the infant population. If confirmed, their use in therapeutic decisions could improve the quality of life and prognosis of these patients.

Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells.

Therapeutic oligonucleotides have achieved great clinical interest since their approval as drug agents by regulatory agencies but their access and distribution in blood cells are not completely known. We evaluated by flow cytometry the ability of short fluorescent scramble oligonucleotides (ON*) to access human peripheral blood mononuclear cells (PBMC) after incubating with ON* during 1 h and 7 days of culture follow-up 'in vitro'. Blood samples were treated with chemically modified oligonucleotides (phosphorothioate backbone and 2' O-Me ends) to resist nuclease digestion under culture conditions. The ON* internalization was determined after discarding the membrane-associated fluorescence by trypan blue quenching. Whereas the oligonucleotide accessed neutrophils and monocytes rapidly, achieving their maximum in 1 h and 24 h, respectively, lymphocytes required 7 days to achieve the maximum (80% of cells) transfection. The ON*ability to access lymphocyte types (T, B, and NK) and T cell subtypes (CD4+, CD8+, and CD4-CD8-) were similar, with T cells being more accessible. Regulatory CD4+ and CD8+ T cells were classified in low and high Foxp3 expressers, whose expression proved not to alter the ON* internalization during the first hour, achieving 53% of CD4+Foxp3+ and 40% of CD8+Foxp3+ cells. Our results contribute to understanding and improving the management of therapeutic ONs.

Pharmacogene Variants Associated with Liver Transplant in a Twelve-Year Clinical Follow-Up.

Some gene polymorphisms have been previously associated individually with tacrolimus efficacy and toxicity, but no long-term study to determine the role of pharmacogene variants in the clinical evolution of liver-transplanted patients has been addressed so far. In the present work, we analyzed the relation between highly-evidenced genetic polymorphisms located in relevant pharmacogenes and the risk of suffering premature death and other comorbidities such as cancer, diabetes mellitus, arterial hypertension, graft rejection, infections and nephrotoxicities in a cohort of 87 patients (8 were excluded due to early loss of follow-up) transplanted at Hospital La Fe in Valencia (Spain) during a 12-year follow-up. Employing a logistic regression model with false discovery rate penalization and Kaplan-Meier analyses, we observed significant association between survival rates and metabolizer genes. In this sense, our results show an association between MTHFR gene variants in donor rs1801133 (HR: 7.90; p-value: 0.032) and recipient rs1801131 (HR: 7.34; p-value: 0.036) and the group of patients who died during the follow-up period, supporting the interest of confirming these results with larger patient cohorts. In addition, donor polymorphisms in UGT1A9 metabolizer gene rs6714486 (OR: 0.13; p-value: 0.032) were associated with a lower risk of suffering from de novo cancer. Genetic variants in CYP2B6 metabolizer gene rs2279343 demonstrated an association with a risk of infection. Other variants in different locations of SLCO1A2, ABCC2 and ABCB1 transporter genes were associated with a lower risk of suffering from type 2 diabetes mellitus, chronic and acute nephrotoxicities and arterial hypertension. Results suggest that pharmacogenetics-derived information may be an important support for personalized drug prescription, clinical follow-up and the evolution of liver-transplanted patients.

Pharmacogenetics in Neuroblastoma: What Can Already Be Clinically Implemented and What Is Coming Next?

Pharmacogenetics is one of the cornerstones of Personalized Precision Medicine that needs to be implemented in the routine of our patients' clinical management in order to tailor their therapies as much as possible, with the aim of maximizing efficacy and minimizing toxicity. This is of great importance, especially in pediatric cancer and even more in complex malignancies such as neuroblastoma, where the rates of therapeutic success are still below those of many other types of tumors. The studies are mainly focused on germline genetic variants and in the present review, state of the art is presented: which are the variants that have a level of evidence high enough to be implemented in the clinic, and how to distinguish them from the ones that still need validation to confirm their utility. Further aspects as relevant characteristics regarding ontogeny and future directions in the research will also be discussed.

Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii.

BACKGROUND: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2'OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. METHODS: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. CONCLUSION: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.

Multicompartmental Lipopolyplex as Vehicle for Antigens and Genes Delivery in Vaccine Formulations.

Vector design and its characterization is an area of great interest in current vaccine research. In this article, we have formulated and characterized a multicompartmental lipopolyplex, which associates multiple liposomes and polyplexes in the same complex. These particles allow the simultaneous delivery of lipid or water-soluble antigens associated with genes to the same cell, in much higher amounts than conventional lipopolyplexes. The vector characterization and optimization were carried out using liposomes with entrapped carboxyfluorescein and adapted electrophoretic assays. Two types of lipopolyplexes (containing hydrophilic or lipophilic antigens) were employed to evaluate their interest in vaccination. The lipopolyplex loaded with an extract of water-soluble melanoma proteins proved to efficiently induce humoral response in murine melanoma model, increasing the levels of IgM and IgG. The specificity of the immune response induced by the lipopolyplex was demonstrated in mice with the lipopolyplex containing the GD3 ganglioside lipid antigen, abundant in melanoma cells. The levels of anti-GD3 IgG increased markedly without modifying the expression of humoral antibodies against other gangliosides.

Mitochondrial DNA Replacement Techniques to Prevent Human Mitochondrial Diseases.

Background: Mitochondrial DNA (mtDNA) diseases are a group of maternally inherited genetic disorders caused by a lack of energy production. Currently, mtDNA diseases have a poor prognosis and no known cure. The chance to have unaffected offspring with a genetic link is important for the affected families, and mitochondrial replacement techniques (MRTs) allow them to do so. MRTs consist of transferring the nuclear DNA from an oocyte with pathogenic mtDNA to an enucleated donor oocyte without pathogenic mtDNA. This paper aims to determine the efficacy, associated risks, and main ethical and legal issues related to MRTs. Methods: A bibliographic review was performed on the MEDLINE and Web of Science databases, along with searches for related clinical trials and news. Results: A total of 48 publications were included for review. Five MRT procedures were identified and their efficacy was compared. Three main risks associated with MRTs were discussed, and the ethical views and legal position of MRTs were reviewed. Conclusions: MRTs are an effective approach to minimizing the risk of transmitting mtDNA diseases, but they do not remove it entirely. Global legal regulation of MRTs is required.

Gold Nanoparticle-Assisted Virus Formation by Means of the Delivery of an Oncolytic Adenovirus Genome.

Oncolytic adenoviruses are a therapeutic alternative to treat cancer based on their ability to replicate selectively in tumor cells. However, their use is limited mainly by the neutralizing antibody (Nab) immune response that prevents repeated dosing. An alternative to facilitate the DNA access to the tumor even in the presence of anti-viral Nabs could be gold nanoparticles able to transfer DNA molecules. However, the ability of these nanoparticles to carry large DNA molecules, such as an oncolytic adenovirus genome, has not been studied. In this work, gold nanoparticles were functionalized with different amounts of polyethylenimine to transfer in a safe and efficient manner a large oncolytic virus genome. Their transfer efficacy and final effect of the oncolytic virus in cancer cells are studied. For each synthesized nanoparticle, (a) DNA loading capacity, (b) complex size, (c) DNA protection ability, (d) transfection efficacy and (e) cytotoxic effect were studied. We observed that small gold nanoparticles (70-80 nm in diameter) protected DNA against nucleases and were able to transfect the ICOVIR-15 oncolytic virus genome encoded in pLR1 plasmid. In the present work, efficient transgene RNA expression, luciferase activity and viral cytopathic effect on cancer cells are reported. These results suggest gold nanoparticles to be an efficient and safe vector for oncolytic adenovirus genome transfer.

MTHFR and VDR Polymorphisms Improve the Prognostic Value of MYCN Status on Overall Survival in Neuroblastoma Patients.

Single nucleotide polymorphisms (SNPs) in Pharmacogenetics can play an important role in the outcomes of the chemotherapy treatment in Neuroblastoma, helping doctors maximize efficacy and minimize toxicity. Employing AgenaBioscience MassArray, 96 SNPs were genotyped in 95 patients looking for associations of SNP with response to induction therapy (RIT) and grade 3-4 toxicities, in High Risk patients. Associations of SNPs with overall (OS) and event-free (EFS) survival in the whole cohort were also explored. Cox and logistic regression models with Elastic net penalty were employed. Association with grade 3-4 gastrointestinal and infectious toxicities was found for 8 different SNPs. Better RIT was correlated with rs726501 AG, rs3740066 GG, rs2010963 GG and rs1143684 TT (OR = 2.87, 1.79, 1.23, 1.14, respectively). EFS was affected by rs2032582, rs4880, rs3814058, rs45511401, rs1544410 and rs6539870. OS was influenced by rs 1801133, rs7186128 and rs1544410. Remarkably, rs1801133 in MTHFR (p = 0.02) and rs1544410 in VDR (p = 0.006) also added an important predictive value for OS to the MYCN status, with a more accurate substratification of the patients. Although validation studies in independent cohorts will be required, the data obtained supports the utility of Pharmacogenetics for predicting Neuroblastoma treatment outcomes.

Progress in the Use of Antisense Oligonucleotides for Vaccine Improvement.

: Antisense oligonucleotides (ASOs) are synthetically prepared short single-stranded deoxynucleotide sequences that have been validated as therapeutic agents and as a valuable tool in molecular driving biology. ASOs can block the expression of specific target genes via complementary hybridization to mRNA. Due to their high specificity and well-known mechanism of action, there has been a growing interest in using them for improving vaccine efficacy. Several studies have shown that ASOs can improve the efficacy of vaccines either by inducing antigen modification such as enhanced expression of immunogenic molecules or by targeting certain components of the host immune system to achieve the desired immune response. However, despite their extended use, some problems such as insufficient stability and low cellular delivery have not been sufficiently resolved to achieve effective and safe ASO-based vaccines. In this review, we analyze the molecular bases and the research that has been conducted to demonstrate the potential use of ASOs in vaccines.

Efficacy of interleukin 10 gene hydrofection in pig liver vascular isolated 'in vivo' by surgical procedure with interest in liver transplantation.

AIM: Liver transplantation is the only curative strategy for final stage liver diseases. Despite the great advances achieved during the last 20 years, the recipient immune response after transplantation is not entirely controlled. This results in high rates of acute cell rejection and, approximately, 10% of early mortality. Therapeutic treatment could be improved by efficiently transfecting genes that encode natural immunosuppressant proteins, employing safe procedures that could be transferred to clinical setting. In this sense, interleukin 10 plays a central role in immune tolerance response by acting at different levels. METHODS: hIL10 gene was hydrofected by retrograde hydrodynamic injection in pig liver with complete vascular exclusion mediated by an 'in vivo' surgical procedure. Levels of IL10 DNA, RNA and protein were determined within liver tissue 1 and 10 days after the injection and, more frequently, also the interleukin-10 protein in peripheral blood. RESULTS: The procedure was safe for the animals and neither hemodynamic parameters nor liver function determinations showed relevant alterations. The hIL10 hydrofection in watertight liver mediated efficient gene transfer and this was transcribed and translated to protein, achieving up to 110 pg/ml of IL10 in peripheral blood. This value is close to that considered able to reduce the activity of TNFα by half (IL10 IC50 for TNFα = 124 pg/ml). CONCLUSIONS: Results of this work suggest that IL10 liver hydrofection with vascular exclusion in vivo is a safe and transferable procedure that mediates plasma protein levels with potential clinical interest in immune modulation after transplantation.

Colorectal cancer: pharmacogenetics support for the correct drug prescription.

Pharmacogenetics (PGx) in clinical practice is a tool that the clinician can use to guide, in a personalized way, the most suitable treatment that will be administered to the patient. The objective of this review is to summarize in a practical and accessible rational way, the advances that currently exist for the application of PGx in colorectal cancer chemotherapy management through the study of the patients' germline polymorphisms. To define the polymorphisms that can be applied, we rely on three fundamental cornerstones: the recommendations of drug regulatory agencies; the implementation guidelines prepared by expert consortia in PGx and information from clinical annotations (the drug/polymorphism relation) according to the scientific level of evidence assigned by PharmGKB experts.

Pharmacogenetics implementation in the clinics: information and guidelines for germline variants.

The aim of this work was to supply an overview of the germline Pharmacogenetics that can be already implemented in the oncology clinical practice. An explanation of the three pillars considered necessary for determining which genetic polymorphisms should be used has been provided. These are PharmGKB single nucleotide polymorphism (SNP)-Drug Clinical Annotations with levels of evidence 1 or 2; the genetic information provided in the drug labels by the drug regulatory main agencies (Food and Drug Administration and European Medicines Agency, mainly); and the guidelines elaborated by international expert consortia (mainly Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacogenetics Working Group). A summary of the relevant SNPs and the recommendations on how to apply their results has also been compiled.

Translational Advances of Hydrofection by Hydrodynamic Injection.

Hydrodynamic gene delivery has proven to be a safe and efficient procedure for gene transfer, able to mediate, in murine model, therapeutic levels of proteins encoded by the transfected gene. In different disease models and targeting distinct organs, it has been demonstrated to revert the pathologic symptoms and signs. The therapeutic potential of hydrofection led different groups to work on the clinical translation of the procedure. In order to prevent the hemodynamic side effects derived from the rapid injection of a large volume, the conditions had to be moderated to make them compatible with its use in mid-size animal models such as rat, hamster and rabbit and large animals as dog, pig and primates. Despite the different approaches performed to adapt the conditions of gene delivery, the results obtained in any of these mid-size and large animals have been poorer than those obtained in murine model. Among these different strategies to reduce the volume employed, the most effective one has been to exclude the vasculature of the target organ and inject the solution directly. This procedure has permitted, by catheterization and surgical procedures in large animals, achieving protein expression levels in tissue close to those achieved in gold standard models. These promising results and the possibility of employing these strategies to transfer gene constructs able to edit genes, such as CRISPR, have renewed the clinical interest of this procedure of gene transfer. In order to translate the hydrodynamic gene delivery to human use, it is demanding the standardization of the procedure conditions and the molecular parameters of evaluation in order to be able to compare the results and establish a homogeneous manner of expressing the data obtained, as 'classic' drugs.

Hydrodynamic IL10 Gene Transfer in Human Colon: Results from an "EX VIVO" Study with Potential Clinical Application in Crohn's Disease.

BACKGROUND: The aim of this work is to evaluate the efficacy of hydrodynamic venous IL10 gene delivery to "ex vivo" human colon segments and to determine its potential interest in Crohn's disease treatment. METHODS: Twenty human colon segments were obtained from surgical resections. Hydrodynamic transfection through the main vein of the pedicle with 50 mL of hIL10 plasmid (20 µg/mL) solution was performed on 13 of them. Tissue sections were cultured and DNA, RNA, and protein copies were determined after 1, 2, and 4 days. Data obtained were compared with 6 nontransfected specimens. Finally, 1 specimen was injected with gold nanoparticles, and their distribution was examined under electron microscope. RESULTS: IL10 DNA levels were higher in treated tissues than in controls (P < 0.001), decreasing along time. The amount of hIL10 RNA was significantly increased in treated tissues when compared with controls (P = 0.001). The indexes of protein IL10 translation in treated groups were much higher (P < 0.001) than the basal production. The protein expression was higher in transfected tissue (10-50-fold, with respect to control tissue); this difference being established during the first hours and maintained during, at least, 4 days. With electron microscopy, we hardly observed large (15 nm) gold nanoparticles within the tissue, always in the submucosa. However, multiple small (4 nm) nanoparticles were observed within the cytoplasm of enterocytes in mucosa. CONCLUSIONS: Hydrodynamic procedure efficiently delivers the IL10 gene to the human colon, achieving levels of tissue protein expression high enough to mediate pharmacological effects with interest in controlling immune response in patients with Crohn's disease.

Influence of cytarabine metabolic pathway polymorphisms in acute myeloid leukemia induction treatment.

Cytarabine is considered the most effective chemotherapeutic option in acute myeloid leukemia (AML). The impact of 10 polymorphisms in cytarabine metabolic pathway genes were evaluated in 225 adult de novo AML patients. Variant alleles of DCK rs2306744 and CDA rs602950 showed higher complete remission (p = .024, p = .045), with lower survival rates for variant alleles of CDA rs2072671 (p = .015, p = .045, p = .032), rs3215400 (p = .033) and wild-type genotype of rs602950 (p = .039, .014). Induction death (p = .033) and lower survival rates (p = .021, p = .047) were correlated to RRM1 rs9937 variant allele. In addition, variant alleles of CDA rs532545 and rs602950 were related to skin toxicity (p = .031, p = .049) and mucositis to DCK rs2306744 minor allele (p = .046). Other toxicities associated to variant alleles were hepatotoxicity to NT5C2 rs11598702 (p = .032), lung toxicity (p = .031) and thrombocytopenia to DCK rs4694362 (p = .046). This study supports the interest of cytarabine pathway polymorphisms regarding efficacy and toxicity of AML therapy in a coherent integrated manner.

Silencing of Foxp3 enhances the antitumor efficacy of GM-CSF genetically modified tumor cell vaccine against B16 melanoma.

The antitumor response after therapeutic vaccination has a limited effect and seems to be related to the presence of T regulatory cells (Treg), which express the immunoregulatory molecules CTLA4 and Foxp3. The blockage of CTLA4 using antibodies has shown an effective antitumor response conducing to the approval of the human anti-CTLA4 antibody ipilimumab by the US Food and Drug Administration. On the other hand, Foxp3 is crucial for Treg development. For this reason, it is an attractive target for cancer treatment. This study aims to evaluate whether combining therapeutic vaccination with CTLA4 or Foxp3 gene silencing enhances the antitumor response. First, the "in vitro" cell entrance and gene silencing efficacy of two tools, 2'-O-methyl phosphorotioate-modified oligonucleotides (2'-OMe-PS-ASOs) and polypurine reverse Hoogsteen hairpins (PPRHs), were evaluated in EL4 cells and cultured primary lymphocytes. Following B16 tumor transplant, C57BL6 mice were vaccinated with irradiated B16 tumor cells engineered to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) and were intraperitoneally treated with CTLA4 and Foxp3 2'-OMe-PS-ASO before and after vaccination. Tumor growth, mice survival, and CTLA4 and Foxp3 expression in blood cells were measured. The following results were obtained: 1) only 2'-OMe-PS-ASO reached gene silencing efficacy "in vitro"; 2) an improved survival effect was achieved combining both therapeutic vaccine and Foxp3 antisense or CTLA4 antisense oligonucleotides (50% and 20%, respectively); 3) The blood CD4+CD25+Foxp3+ (Treg) and CD4+CTLA4+ cell counts were higher in mice that developed tumor on the day of sacrifice. Our data showed that tumor cell vaccine combined with Foxp3 or CTLA4 gene silencing can increase the efficacy of therapeutic antitumor vaccination.

Impact of ABC single nucleotide polymorphisms upon the efficacy and toxicity of induction chemotherapy in acute myeloid leukemia.

Anthracycline uptake could be affected by efflux pumps of the ABC family. The influence of 7 SNPs of ABC genes was evaluated in 225 adult de novo acute myeloid leukemia (AML) patients. After multivariate logistic regression there were no significant differences in complete remission, though induction death was associated to ABCB1 triple variant haplotype (p = .020). The ABCB1 triple variant haplotype was related to higher nephrotoxicity (p = .016), as well as this haplotype and the variant allele of ABCB1 rs1128503, rs2032582 to hepatotoxicity (p = .001; p = .049; p < .001). Furthermore, the variant allele of ABCC1 rs4148350 was related to severe hepatotoxicity (p = .044), and the variant allele of ABCG2 rs2231142 was associated to greater cardiac (p = .004) and lung toxicities (p = .038). Delayed time to neutropenia recovery was observed with ABCB1 rs2032582 variant (p = .047). This study shows the impact of ABC polymorphisms in AML chemotherapy safety. Further prospective studies with larger population are needed to validate these associations.

Studying Closed Hydrodynamic Models of "In Vivo" DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans.

INTRODUCTION: Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest. MATERIAL AND METHODS: A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated. RESULTS: Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (p<0.001) than the reference standards in all cases. CONCLUSION: Hydrofection of hAAT DNA to "in vivo" isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and surgically closed models mediate high tissue protein expression. Impairment of protein secretion to plasma is observed and might be species-related. This study reinforces the potential application of closed liver hydrofection for therapeutic purposes, provided protein secretion improves.