Impact of NADPH oxidase functional polymorphisms in acute myeloid leukemia induction chemotherapy.

Efficacy and toxicity of anthracycline treatment in acute myeloid leukemia (AML) is mediated by reactive oxygen species (ROS). NADPH oxidase is the major endogenous source of ROS and a key mediator of oxidative cardiac damage. The impact of NADPH oxidase polymorphisms (CYBA:rs4673, NCF4:rs1883112, RAC2:rs13058338) was evaluated in 225 adult de novo AML patients. Variant alleles of NCF4 and RAC2 were related to higher complete remission (P=0.035, P=0.016), and CYBA homozygous variant showed lower overall survival with recessive model (P=0.045). Anthracycline-induced cardiotoxicity was associated to NCF4 homozygous variant (P=0.012) and CYBA heterozygous genotype (P=0.027). Novel associations were found between variant allele of CYBA and lower lung and gastrointestinal toxicities, and a protective effect in nephrotoxicity and RAC2 homozygous variant. Moreover, RAC2 homozygous variant was related to delayed thrombocytopenia recovery. This study supports the interest of NADPH oxidase polymorphisms regarding efficacy and toxicity of AML induction therapy, in a coherent integrated manner.

A systematic review and meta-analysis of the impact of WT1 polymorphism rs16754 in the effectiveness of standard chemotherapy in patients with acute myeloid leukemia.

The polymorphism rs16754 of the WT1 gene has been described as a possible prognostic marker in different acute myeloid leukemia (AML) cohorts; however, it is not supported by all the studies. We performed the first meta-analysis evaluating the effect of this polymorphism upon the effectiveness of standard AML therapy. Fourteen cohort studies were included (3618 patients). Patients with the variant allele showed a significant higher overall survival (OS) at 5 years (OR:1.24, 95% CI: 1.06-1.45, P=0.007, with dominant model). WT1 did not influence complete remission, but a higher disease-free survival was observed with the variant allele. In the subgroup analysis, Caucasians, pediatric and patients treated with idarubicin and etoposide carrying the variant allele showed consistent results in OS, whereas patients with cytogenetically normal AML did not show differences. To verify the effect of this polymorphism upon other outcomes, studies in larger and multiracial populations are needed.

Influence of ABCB1 polymorphisms upon the effectiveness of standard treatment for acute myeloid leukemia: a systematic review and meta-analysis of observational studies.

The ABCB1 gene encodes for P-glycoprotein (P-gp), an efflux pump for a variety of xenobiotics. The role of ABCB1 polymorphisms in acute myeloid leukemia (AML) outcomes of standard chemotherapy (cytarabine plus anthracyclines) remains controversial. A systematic search was made of studies evaluating the association between ABCB1 polymorphisms 1236C>T, 2677G>T/A and 3435C>T and effectiveness variables. We found seven cohort studies (1241 patients) showing a significantly higher overall survival (OS) among carriers of the variant allele of 1236C>T at year 4 (odds ratio (OR): 1.47, 95% confidence interval (CI): 1.07-2.01), 2677G>T/A at years 4-5 (OR: 1.37, 95% CI: 1.01-1.86) and 3435C>T at years 3 (OR: 1.41, 95% CI: 1.03-1.94) and 4-5 (OR: 1.42, 95% CI: 1.05-1.91). In the subgroup analysis according to ethnicity, Caucasians carrying variant allele showed consistent results in OS. ABCB1 influence upon complete remission could not be demonstrated. Future studies based on larger populations and multiethnic groups should help clarify the effect of P-gp polymorphisms upon other outcomes.

Effect of CYP3A5*3 on kidney transplant recipients treated with tacrolimus: a systematic review and meta-analysis of observational studies.

The highly variable pharmacokinetics of tacrolimus can hamper the optimal management of kidney transplant patients. This variability has been attributed to the genetic polymorphism of CYP3A5 6986A>G, but the evidence is not clear. We conducted a meta-analysis of studies evaluating the effect of CYP3A5 polymorphism on kidney transplant recipients with tacrolimus plasma concentration divided by daily dose per body weight (C/D) and clinical outcomes. We searched in MEDLINE and EMBASE. We found evidence suggesting a significantly lower C/D among CYP3A5*1 allele carriers compared with carriers of the CYP3A5*3/*3 genotype at weeks 1 and 2, and months 1, 3, 6 and 12. We demonstrated that the expresser genotype might have higher risk of acute rejection and chronic nephrotoxicity. In conclusion, CYP3A5 6986A>G polymorphism can affect tacrolimus pharmacokinetics and the incidence of acute rejection and chronic nephrotoxicity on kidney transplant recipients. Patients at high risk of developing tacrolimus-related complications could be detected even before their kidney transplant.

A surgical model for isolating the pig liver in vivo for gene therapy.

Several studies report results that suggest the need of vascularization blocking for efficient gene transfer to the liver, especially in nonviral gene therapy. In this study, we describe a surgical strategy for in vivo isolation of the pig liver, resulting in a vascular watertight organ that allows the evaluation of several gene injection conditions. The hepatic artery and portal, suprahepatic and infrahepatic cava veins were dissected. Then, liver vascularization was excluded for 5-7 min. In that time, we first injected 200 ml saline solution containing the p3c-eGFP plasmid (20 µg/ml) simultaneously through two different catheters placed in the portal and cava veins, respectively. Vital constants were monitored during the surgery to assess the safety of the procedure. Basal systolic/diastolic blood pressures were 92.8/63.2 mm Hg and dropped to 40.7/31.3 mm Hg at the end of vascular exclusion; the mean basal heart rate was 58 bpm, reaching 95 bpm when the blood pressure was low. Oxygen saturation was maintained above 98% during the intervention, and no relevant changes were observed in the ECG tracing. Peak plasma AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were observed after 24 h (151 and 57 IU, respectively). These values were higher, but not relevant, in 60 ml/s injection than in 20 ml/s injection. Efficiency of gene transfer was studied with simultaneous (cava and portal veins) injection of eGFP gene at flow rates of 20 and 60 ml/s. Liver tissue samples were collected 24 h after injection and qPCR was carried out on each lobe sample. The results confirmed the efficiency of the procedure. Gene delivery differed between 20 ml/s (9.9-31.0 eGFP DNA copies/100 pg of total DNA) and 60 ml/s injections (0.6-1.1 eGFP DNA copies/100 pg of total DNA). Gene transcription showed no significant differences between 20 ml/s (15,701.8-21,475.8 eGFP RNA copies/100 ng of total RNA) and 60 ml/s (12,014-36,371 eGFP RNA copies/100 ng of total RNA). The procedure is not harmful for animals and it offers a wide range of gene delivery options because it allows different perfusion ways (anterograde and retrograde) and different flow rates to determine the optimal conditions of gene transfer. This strategy permits the use of cell therapy and viral or non-viral liver gene therapy, especially appropriated to a wide variety of inherited or acquired diseases because of the liver's ability to produce and deliver proteins to the bloodstream.

Comparative antitumor effect among GM-CSF, IL-12 and GM-CSF+IL-12 genetically modified tumor cell vaccines.

Genetically modified cells have been shown to be one of the most effective cancer vaccine strategies. An evaluation is made of the efficacy of both preventive and therapeutic antitumor vaccines against murine melanoma, using C57BL/6 mice and irradiated B16 tumor cells expressing granulocyte and macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12) or both. Tumor was transplanted by the injection of wild-type B16 cells. Tumor growth and survival were measured to evaluate the efficacy of vaccination. Specific humoral response and immunoglobulin G (IgG) switch were evaluated measuring total IgG and IgG1 and IgG2a subtypes against tumor membrane proteins of B16 cells. In preventive vaccination, all treated groups showed delayed tumor growth. In addition, the group vaccinated to express only GM-CSF achieved 100% animal survival (P<0.005). Vaccination with GM-CSF+IL-12-producing B16 cells yielded lesser results (60% survival, P<0.005). Furthermore, all surviving animals remained disease-free after second tumor implantation 1 year later. The therapeutic vaccination strategies resulted in significantly delayed tumor growth, mainly using B16 cells producing GM-CSF+IL-12 cytokines, with 70% tumor growth inhibition (P<0.001)-although none of the animals reached overall survival. The results obtained suggest that the GM-CSF+IL-12 combination only increases the efficacy of therapeutic vaccines. No differences in classical regulatory T cells were found among the different groups.

DNA delivery to 'ex vivo' human liver segments.

Hydrodynamic injection is an efficient procedure for liver gene therapy in rodents but with limited efficacy in large animals, using an 'in vivo' adapted regional hydrodynamic gene delivery system. We study the ability of this procedure to mediate gene delivery in human liver segments obtained by surgical resection. Watertight liver segments were retrogradely injected from hepatic vein with a saline solution containing a plasmid bearing the enhanced green fluorescent protein (eGFP) gene, under different conditions of flow rate (1, 10 and 20 ml s(-1)) and final perfused volume. Samples were cultured for 1 to 2 days and used for microscopy and molecular analysis of gene expression. The fluorescent and immunohistochemistry studies indicated that in segments injected at ≥10 ml s(-1), good and wide gene expression was present in the liver sections and the molecular analysis reinforced the histological observation in a quantitative manner (index of apparent gene delivery: 10(2)-10(4) eGFP DNA copy per 100 pg of total DNA; transcription index: 10(5)-2 × 10(6) eGFP RNA copy per 100 ng of total RNA). In addition, injected gold nanoparticles (15 nm diameter) suggested that DNA delivery to hepatocytes must involve a facilitated permeation process without membrane disruption. In summary, we show that retrograde venous injection of watertight human liver segment is an anadromous procedure that results in wide liver gene delivery and good gene expression. However, additional studies will be necessary to clarify the influence of the prolonged ischemia injury to hepatocytes in our model.

Influence of pharmacogenetic polymorphisms in routine immunosuppression therapy after renal transplantation.

Pharmacogenetics is the study of the cause of various individual responses to the same pharmacologic therapy. Genetic alterations in a single nucleotide in the genes responsible for transport and metabolism of an immunosuppression drug may modify patient response. Although pharmacogenetics is of interest, its clinical relevance remains to be demonstrated. The objective of the present study was to evaluate the effect of single-nucleotide polymorphisms (SNPs) in renal transplant recipients and their donors relative to blood concentrations of tacrolimus in the first 2 weeks posttransplantation. Seventy-one blood samples each from renal transplant recipients and their donors were analyzed using a genetic analysis system (MassARRAY; Sequenom, Inc, San Diego, California) in an attempt to characterize the more relevant SNPs of the ABCB1 and CYP3A5 genes for correlation with recipient trough concentrations of drug. Two-way analysis of variance and Bonferroni post hoc tests were used. In agreement with theoretical predictions, the wild-type genotype in ABCB1 SNPs (CC) tended to stabilize drug concentrations within the therapeutic range, whereas the T variant induced a mean increase in blood concentrations of more than 60%. These findings are in agreement with statistical tests that compared mean concentrations in various recipient-donor populations and found significant differences between them (P<.001) in CC vs TT, and P<.01 in CT vs TT). Donor genotype did not seem to be relevant. However, further studies are required to achieve more robust conclusions.

Clinical interest of pharmacogenetic polymorphisms in the immunosuppressive treatment after heart transplantation.

In the transplantation field, genetic changes in a single nucleotide in the genes responsible for the transport and metabolism of an immunosuppressive drug may modify the response of the patient. The aim of this study was to evaluate the effect of single nucleotide polymorphisms (SNPs) in heart transplant recipients and their donors in association with tacrolimus and cyclosporine blood levels during the first 2 weeks after transplantation. A total of 18 blood samples from heart transplant recipients and their donors (n=36) were analyzed using Sequenom to characterize the more relevant SNPs of the ABCB1 and CYP3A5 genes for correlation with C0 (trough concentration) drug blood levels. Differences between groups were evaluated with two-way analysis of variance (ANOVA) and Bonferroni post-test. In agreement with theoretical predictions, the wild type genotype in ABCB1 SNPs (CC) tended to stabilize drug levels within the therapeutic range, whereas the T variant induced a 79% mean increase in blood levels among heterozygous (CT) and 100% among homozygous (TT) recipients. These results agreed with the mean levels in various recipient/donor populations, finding significant differences between them (P<.001 in CC vs CT and P<.01 in CT vs TT), as well as a certain influence of the donor genotype.

Pig liver gene therapy by noninvasive interventionist catheterism.

The efficacy of noninvasive interventionist catheterism in large animals as an alternative to the hydrodynamic procedure, described for small animals, is evaluated. Basically, gene transfer is performed by implantation and fixation of a balloon catheter within the suprahepatic vein of anesthetized pigs, through the femoral vein. The catheter tip is identified by fluoroscopy, injecting a contrast solution that marks large or small hepatic territories. Animals were injected with a 100 ml pTG7101 plasmid solution (40 microg/ml), which contains the human alpha-1 antitrypsin gene, perfused at a rate of 7.5 ml/s and efficacy and toxicity of the procedure were evaluated. The results show: (i) the highest efficacy in protein production is reached when perfusion is limited to small areas of the liver; (ii) no relevant hepatic toxicity was observed; (iii) gene transfer is mainly located in the areas around the central vein, as seen in the immunohistochemical studies; (iv) the electron microscopy studies indicate that the areas with good transfection efficacy show the presence of abundant endocytic vesicles that may even fuse among themselves. These data suggest that retrovenous injection by noninvasive interventionist catheterism could become an efficient procedure for hepatic gene transfer with potential clinical applications.

Real-time quantification of human telomerase reverse transcriptase mRNA in the plasma of patients with prostate cancer.

The aim of this study was to evaluate the potential diagnostic value of quantitative analysis of human telomerase reverse transcriptase (hTERT) mRNA in plasma for noninvasive diagnosis of prostate cancer (PCa). Expression levels of hTERT were analyzed by real-time quantitative RT-PCR in 68 patients showing elevated prostate-specific antigen (PSA) levels and a control group of 44 healthy volunteers. Sensitivity and specificity were determined and compared to the corresponding PSA values. Median values for hTERT gene expression in the PCa patients (0.72 ng; range 0.01-12.86) were statistically significantly higher (P < 0.001) than in the control group (0.13 ng; 0.02-0.35). Patients with clinically confirmed prostatitis showed lower plasma hTERT expression than PCa patients (0.29; 0.01-66.07). At a cutoff value of 0.35 sensitivity and specificity for the diagnosis of PCa were 81% and 60%, respectively. We suggest that hTERT mRNA in plasma is a very specific and sensitive method that may aid to differentiate between malignant and nonmalignant prostate tissue and may be a useful marker (in combination with PSA) for early PCa diagnosis.

Antisense gene therapy using anti-k-ras and antitelomerase oligonucleotides in colorectal cancer.

AIM: To test the efficacy of anti-k-ras and antitelomerase oligonucleotides for disabling colorectal cancer cell growth. MATERIAL AND METHODS: An established human colorectal cancer cell line (SW 480, ATTC) was used. Oligodeoxiribonucleotides (ODNs) have a phosphorotioate modification to ensure intracellular intake. We used an antitelomerase ODN (Telp5) and two anti-k-ras ODNs (AS-KRAS and ISIS). AS-KRAS is designed to join the k-ras oncogene s exon 1. ISIS links to the terminal transcription unit 5 of k-ras. Telp5 joins the template region of the hTR telomerase subunit. ODNs have been tested in different concentrations (1, 5, 10, 20 micromolar). Cell viability has been tested at 48 and 72 hours. Statistical analysis and graphic design were made with the statistical package "Analyzing Data with GraphPad Prism-1999", GraphPad Sofware Inc., San Diego CA. We used the Student's t test for statistical analysis. RESULTS: The lowest dose (1 microM) was not effective. Using the highest dose (20 microM for 48 hours) of combined AS-KRAS and Telp5 cell viability decreased to 99.67%. The rest of results varied depending on ODN type, dose, and exposure time. CONCLUSIONS: Tested antisense ODNs stop colorectal cancer cell growth, and a combination of anti-telomerase and anti-k-ras is the most useful treatment. Efficacy is best with a higher dose and longer treatment period.

Eficacia de la terapia génica antisentido utilizando oligonucleótidos anti K-ras y antitelomerasa en cáncer colorrectal / Antisesnse gene therapy using anti-k-ras and antitelomerase oligonucleotides in colorectal cancer

Objetivo: evaluar la eficacia de oligonucleótidos anti k-ras yantitelomerasa para detener el crecimiento tumoral en el cáncercolorrectal.Material y métodos: se ha empleado una línea celular establecidade cáncer colorrectal humano (SW 480, ATTC®). Los oligodesoxirribonucleótidos(ODN) utilizados en el presente trabajo presentanmodificación fosforotioato con el fin de mejorar su estabilidad enpresencia de fluidos biológicos. Hemos utilizado un ODN antitelomerasa(Telp5), y dos ODN anti k-ras (AS-KRAS e ISIS). AS-KRAS actúaen el exón 1 e ISIS actúa a nivel de la unidad terminal de transcripción5’ del oncogen k-ras. Telp5 se une a la subunidad hTR de latelomerasa. Se han aplicado en concentraciones 1, 5, 10 y 20 micromolar,midiendo la viabilidad celular a las 48 y 72 horas de tratamiento.El análisis estadístico y el diseño de los gráficos se han realizadomediante el programa “Analyzing Data with GraphPadPrism-1999”. GraphPad Sofware Inc., San Diego CA©. Para el tratamientoestadístico se ha utilizado el test t de Student.Resultados: la dosis mínima (1 µM) no fue efectiva ni a 48 nia 72 horas postratamiento. Con la dosis máxima (20 µM durante48 horas) y utilizando la combinación de AS-KRAS y Telp5 obtuvimosuna reducción de la viabilidad celular del 99,67%. El restode resultados fueron intermedios, dependiendo del tipo de oligonucleótidoempleado, la dosis y el tiempo de exposición.Conclusiones: los oligonucleótidos antisentido probados detienenel crecimiento celular en el cáncer colorrectal, siendo larespuesta más eficaz la combinación de ambos y aumentando dichaeficacia con mayor dosis y tiempo de exposición

Aim: to test the efficacy of anti-k-ras and antitelomeraseoligonucleotides for disabling colorectal cancer cell growth.Material and methods: an established human colorectalcancer cell line (SW 480, ATTC®) was used. Oligodeoxiribonucleotides(ODNs) have a phosphorotioate modification to ensureintracellular intake. We used an antitelomerase ODN (Telp5) andtwo anti-k-ras ODNs (AS-KRAS and ISIS). AS-KRAS is designedto join the k-ras oncogene’s exon 1. ISIS links to the terminaltranscription unit 5’ of k-ras. Telp5 joins the template region ofthe hTR telomerase subunit. ODNs have been tested in differentconcentrations (1, 5, 10, 20 micromolar). Cell viability has beentested at 48 and 72 hours. Statistical analysis and graphic designwere made with the statistical package “Analyzing Data withGraphPad Prism-1999”, GraphPad Sofware Inc., San DiegoCA©. We used the Student’s t test for statistical analysis.Results: the lowest dose (1 µM) was not effective. Using thehighest dose (20 µM for 48 hours) of combined AS-KRAS andTelp5 cell viability decreased to 99.67%. The rest of results varieddepending on ODN type, dose, and exposure time.Conclusions: tested antisense ODNs stop colorectal cancercell growth, and a combination of anti-telomerase and anti-k-ras isthe most useful treatment. Efficacy is best with a higher dose andlonger treatment period

Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.

The present study contributes to clarify the mechanism underlying the high efficacy of hepatocyte gene transfer mediated by hydrodynamic injection. Gene transfer experiments were performed employing the hAAT gene, and the efficacy and differential identification in mouse plasma of human transgene versus mouse gene was assessed by ELISA and proteomic procedures, respectively. By applying different experimental strategies such as cumulative dose-response efficacy, hemodynamic changes reflected by venous pressures, intravital microscopy, and morphological changes established by transmission electron microscopy, we found that: (a) cumulative multiple doses of transgene by hydrodynamic injection are efficient and well tolerated, resulting in therapeutic plasma levels of hAAT; (b) hydrodynamic injection mediates a transient inversion of intrahepatic blood flow, with circulatory stasis for a few minutes mainly in pericentral vein sinusoids; (c) transmission electron microscopy shows hydrodynamic injection to promote massive megafluid endocytic vesicles among hepatocytes around the central vein but not in hepatocytes around the periportal vein. We suggest that the mechanism of hydrodynamic liver gene transfer involves transient inversion of intrahepatic flow, sinusoidal blood stasis, and massive fluid endocytic vesicles in pericentral vein hepatocytes.

Real time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA in patients with colorectal cancer.

BACKGROUND: Increased telomerase activity can be found in almost 90% of colorectal tumours. We aim to describe the preliminary results for quantification in plasma of hTERT mRNA in colorectal cancer patients. MATERIALS AND METHODS: Fifty patients undergoing surgery for colorectal cancer and a control group of 50 healthy volunteers were prospectively studied. Pre-operative venous blood samples were taken from all cancer patients and volunteers. Plasma hTERT expression was determined from peripheral blood based on real-time quantitative RT-PCR (qRT-PCR) method normalized to the amount of RNA input using 18S rRNA gene expression. Plasma pre-operative CEA levels were also determined. RESULTS: Median values for normalized hTERT (hTERT(N)) gene expression were higher in colorectal cancer patients (11.62, range 0.23-47.67) than healthy volunteers (0.29, range 0.00-4.63) (P < 0.001). Individual data showed that 82% of colorectal cancer patients had hTERT(N) expression values superior to the maximum value observed in the control group. Sensitivity and specificity of the assay for colorectal cancer detection were 98% and 64%, respectively. No significant differences in hTERT(N) expression between gender or with age (P > 0.05). No significant correlation was found between hTERT(N) expression and CEA values (Spearman's rank test = 0.136, P = 0.348). CONCLUSIONS: These results show that detection of mRNA based on the qRT-PCR of the telomerase hTERT(N) gene in plasma clearly differentiates between healthy and colorectal cancer patients and that hTERT(N) can be detected and quantified in plasma. This opens up a new field as a noninvasive blood test for colorectal cancer diagnosis.

Long-term therapeutic levels of human alpha-1 antitrypsin in plasma after hydrodynamic injection of nonviral DNA.

The transfection efficacy of several vectors containing the full genomic hAAT gene with its natural promoter (pTG7101) and others containing the cDNA of hAAT gene driven by cytomegalovirus immediate-early promoter or the 0.5 kb upstream of hAAT gene sequence has been studied by hydrodynamic tail-vein injection (20 microg/mouse). pTG7101 (but not the other plasmids) results in therapeutic and stable concentration of hAAT in plasma. A dose-response study with this plasmid (0.3-320 microg/mouse) confirms that hAAT remains long-term stable in plasma, with therapeutic concentrations of hAAT (>0.9 mg/ml). The parameters of the dose-response curve were: R: 0.98, E(max) 3449.0+/- 279.7 microg/ml and EC(50) 1.2 x 10(12) plasmid-gene units. In addition, 4 months after transfection, the intrinsic efficacy of transgenic expression (amount of RNA/DNA) in mouse liver was 50-80% that normally expressed by the mouse gene. The important efficacy of nonviral genomic DNA opens a new avenue in the safety applications of human gene therapy.

Stability of PEI-DNA and DOTAP-DNA complexes: effect of alkaline pH, heparin and serum.

DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) are widely used in gene therapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures for DNA quantification from DNA complexes as well as their stability in the presence of DNase or mouse, rat and human sera. Release of the DNA from its complexes was accomplished by increasing the pH of the medium (from 7.3 to 13.4) or by adding heparin. The stability against degradation was tested in vitro, by incubating the complexes at 37 degrees C in the presence of DNase I and sera from the three species. Both high pH and heparin were able to release DNA from its complexes. Naked DNA formed aggregates with serum proteins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA from digestion by serum DNase, and the DNA digesting ability of serum was: mouse>rat>human. The DNA from the complexes was resistant to degradation by DNase I, although a low proportion of DNA from the complexes was partially digested, as determined by electrophoresis. In contrast, PEI-DNA and DOTAP-DNA complexes were stable in the presence of all sera. Heparin and high pH release DNA from its complexes. The order of DNA degradation is: mouse>rat>human, but DOTAP and PEI avoid degradation of DNA by serum compounds.

Asialofetuin liposome-mediated human alpha1-antitrypsin gene transfer in vivo results in stationary long-term gene expression.

The development of nonviral vectors for in vivo gene delivery to hepatocytes is an interesting topic in view of their safety and tremendous gene therapy potential. Since cationic liposomes and liposome uptake by receptor-mediated mechanisms could offer advantages in the efficacy of liposome-mediated gene transfer, we studied the effect of liposome charge (anionic vs. cationic) and the covalently coupled asialofetuin ligand on the liposome surface in mediating human alpha1-antitrypsin (hAAT) gene transfer to mice in vivo. The changes in liposome charge were made by adding the following lipids to the backbone liposomes: anionic phosphatidylserine, cationic N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or a lipopeptide synthesized from dipalmitoylphosphatidylethanolamine and covalently coupled to the cationic nuclear localization signal peptide. Two plasmids containing the hAAT gene were used: pTG7101, containing the complete genomic sequence of the human gene driven by the natural promoter, and p216, containing the human hAAT cDNA under the control of the CMV promoter. The results indicate that both untargeted anionic and cationic liposomes mediate plasma levels of hAAT that decline over time. However, asialofetuin liposomes increase the plasma levels of hAAT and can mediate long-term gene expression (>12 months) with stationary plasma levels of protein. Results from quantitative and qualitative reverse transcriptase polymerase chain reaction match those from protein plasma levels and confirm both the human origin of the message and the liver as source of the protein. The use of asialofetuin liposomes in hepatic gene therapy may both increase and prolong in vivo gene expression of hAAT and other clinically important genes.

Antitumor effect of B16 melanoma cells genetically modified with the angiogenesis inhibitor rnasin.

The growth of new blood vessels is an essential condition for the development of tumors with a diameter greater than 1-2 mm and also for their metastatic dissemination. RNasin, the placental ribonuclease inhibitor, is known to have antiangiogenic activity through the inhibition of angiogenin and basic fibroblast growth factor. Nevertheless, the administration of the recombinant form of a protein poses several limitations; as a result, we have studied the antitumor effect of RNasin in a murine gene therapy model. RNasin cDNA was subcloned into the pcDNA3 expression vector, and the resulting recombinant plasmid was used to transfect the B16 murine melanoma cell line. An RNasin inverted construction was used as control. Mice intravenously injected with clones expressing RNasin showed a significant inhibition of tumor metastatic progression with respect to control groups (P<.001) and survived longer (P<.001). Tissue sections from RNasin-expressing cell tumors showed a lower number of blood vessels when compared to tissue sections from mice lungs that had been inoculated with control cell lines. The results of these experiments show that the genetic modification of tumor cells with RNasin cDNA yields a significant antitumor effect, and suggest that this effect is at least partially the result of angiogenesis inhibition.