Biotherapies of neuromuscular disorders.

This review focuses on the most recent data on biotherapeutic approaches, using DNA, RNA, recombinant proteins, or cells as therapeutic tools or targets for the treatment of neuromuscular diseases. Many of these novel technologies have now reached the clinical stage and have or are about to move to the market. Others, like genome editing are still in an early stage but hold great promise.

Transplantation of hepatocytes in nonhuman primates: a preclinical model for the treatment of hepatic metabolic diseases.

BACKGROUND: The transplantation of isolated hepatocytes in large animals, including nonhuman primates, must be evaluated before clinical trials are performed. However, in the absence of large transgenic animals and large-animal (as opposed to small-animal) models of genetic deficiencies, it is difficult to evaluate the fate of transplanted hepatocytes, their localization, survival, and function within the parenchyma of the host liver. In this work, we aimed to develop a technique for delivering hepatocytes to the liver of a nonhuman primate and to evaluate their localization and functionality in the short term. METHODS: A 20% hepatectomy was performed in 34 cynomolgus monkeys (Macaca fascicularis) and hepatocytes were isolated. Hepatocytes were labeled in vitro with a recombinant retrovirus expressing the beta-galactosidase gene and returned to the liver by infusion through a portal catheter left in place. Liver biopsies were performed 4 and 7 d after transplantation. RESULTS: Twenty-four monkeys underwent surgery to define the necessary technical adjustments and to optimize conditions. Six monkeys died. The whole protocol, including the transplantation of genetically marked hepatocytes and procurement of liver biopsies, was performed in the remaining 10 monkeys. In eight monkeys, transplanted hepatocytes expressing the beta-galactosidase gene were widely distributed in the portal tracts, sinusoids, and hepatocyte plates of the host liver 4 and 7 d after transplantation. CONCLUSIONS: We have developed an experimental nonhuman primate model for the evaluation of hepatocyte transplantation. We demonstrated the engraftment and functioning of transplanted hepatocytes in the host liver 4 and 7 d after transplantation.

[Allotransplantation in utero and immortalization of primate fetal hepatocytes]. / Allotransplantation in utero et immortalisation d'hépatocytes foetaux de primates.

We are developing cell therapy approaches on non-human primates as a preclinical model for the treatment of hepatic metabolic diseases. In foetuses, the tissues, including liver, are in expansion, which should facilitate hepatocytes engraftment, and the immune system becomes fully mature only after birth. We have set out conditions for isolation of fetal hepatocytes from macaca mulatta at the end of the 2nd trimester of gestation (90-100 days), their cryopreservation and retroviral transduction. Two different routes of administration of hepatocytes were evaluated: the umbilical vein which was deleterious for the foetuses, and the intraparenchymatous injection which was well tolerated by the animals. Administration of hepatocytes into the hepatic parenchyma resulted in microchimerism and allogenic cells were visualized 9 days after transplantation. Another approach has been to immortalize simian foetal hepatocytes using a retroviral vector expressing SV40 Large T flanked by lox sites. A cell line has been established for 2 years, which is not tumorigenic when injected subcutaneously into nude mice and display characteristics of bipotent hepatoblasts, precursors of hepatocytes and biliary cells. After orthotopic transplantation into nude mice via the portal vein, these cells expressed albumin until the sacrifice of the animals (17 days). The next steps will be to define conditions for transplantation of retrovirally transduced fetal primary and/or immortalized hepatocytes into young foetuses (60 days of gestation) and post-natally.

Monitoring cyclical air/water elemental mercury exchange.

Previous experimental work has demonstrated that elemental mercury evasion from natural water displays a diel cycle; evasion rates during the day can be two to three times evasion rates observed at night. A study with polychlorinated biphenyls (PCBs) found that diurnal PCB air/water exchange rates exceeded nocturnal exchange rates by 32%. Given that the exchange rates of both PCBs and elemental mercury are dominated by the resistance in the aqueous thin film at the air/water interface and that water column elemental mercury concentrations in natural water bodies also display a diel cycle (and water column PCB concentrations do not) the findings here suggest that PCBs can serve as a tracer to assess the relative contribution of diel atmospheric temperature variations on elemental mercury air/water exchange rates. Using previously published data describing water column elemental mercury concentrations and the previously published diel mercury evasion model, four evasion scenarios are examined within the context of monitoring air/water toxicant exchange: constant atmospheric temperatures and constant water column elemental mercury concentrations; variable atmospheric temperatures and constant water column elemental mercury concentrations; constant atmospheric temperatures and variable water column elemental mercury concentrations; and variable atmospheric temperatures and variable water column elemental mercury concentrations. A scenario of monthly elemental mercury air/water exchange also is examined (at constant atmospheric and water column elemental mercury concentrations). Some of the findings include: (1) atmospheric temperature variations do have a significant effect on air/water toxicant exchange; (2) diel atmospheric temperature variations become more significant to overall diel toxicant exchange rates the closer the air/water system is to equilibrium conditions; (3) for refractory toxicants, average diel exchange rates are best estimated by averaging datasets obtained over a 24 h period or, at minimum, by measuring exchange rates at average atmospheric temperature values; (4) for elemental mercury, variable diel water column concentrations are likely to be the dominant contributor to variations in diel evasion rates; (5) diel atmospheric temperature variations amplify the magnitudes of both diel mercury evasion and absorption events and can shift maximum evasion rates to later in the day; (6) variations in monthly elemental mercury air/water exchange rates may exceed diel variations: and (7) 24 h and monthly monitoring efforts will likely be required to accurately describe diel and annual elemental mercury air/water exchange in a given system.

Engraftment of autologous retrovirally transduced hepatocytes after intraportal transplantation into nonhuman primates: implication for ex vivo gene therapy.

The main impediment to effective ex vivo liver gene therapy of metabolic diseases is the lack of experimental work on large animals to resolve such important issues as effective gene delivery, cell-processing techniques, and the development of appropriate vectors. We have used a nonhuman primate, as a preclinical model, to analyze the limiting steps of this approach using recombinant retroviruses. Seven monkeys (Macaca fascicularis) underwent the complete protocol: their left liver lobe was resected, a catheter was placed in the inferior mesenteric vein and connected to an infusion chamber, and the hepatocytes were isolated, cultured, and transduced with a retroviral vector containing the beta-galactosidase gene. The hepatocytes were harvested and returned to the host via the infusion chamber. Biopsies were taken 4-40 days later. No animal was killed in the course of the experiments. They all tolerated the procedure well. We have developed and defined conditions that permit the proliferation and transduction of up to 90% of the plated hepatocytes. A significant proportion of genetically modified cells, representing up to 3% of the liver mass, were safely delivered to the liver via the chamber. Polymerase chain reaction analysis detected integrated viral DNA sequences and quantitative analysis of the in situ beta-Gal-expressing hepatocytes indicated that a significant amount of transduced hepatocytes, up to 2%, had become integrated into the liver and were functional. These results represent substantial advances in the development of the ex vivo approach and suggest that this approach is of clinical relevance for liver-directed gene therapy.

[Effect of portal branch ligation on liver regeneration in the rat]. / Effet de la ligature portale partielle sur la régénération hépatique chez le rat.

GOAL: The aim of this study was to assess liver regeneration after partial portal ligation. METHODS: 70% partial portal occlusion was obtained by ligation of the left portal vein branch. Total liver weight ratio were measured 96 hours after partial portal occlusion and in sham operated animals. The kinetics of hepatocytes division was evaluated by measuring the incorporation of 5-bromo-21-deoxyuridine into replicating cells at various time points by immunohistochemistry. RESULTS: Partial portal occlusion did not alter the total liver weight 96 hours after surgery. It resulted in atrophy of the ligated lobes and hypertrophy of the lobes with preserved portal flow. Hypertrophy was associated to an increase of the percentage of replicating hepatocytes. The replication rate was maximum at 28 hours with a peak at 12.5% and was prolonged beyond the 48th hour. CONCLUSIONS: Partial portal occlusion results in major and prolonged regeneration process in the liver lobes with preserved portal flow.

In utero allotransplantation of retrovirally transduced fetal hepatocytes in primates: feasibility and short-term follow-up.

In utero allotransplantation of fetal hepatocytes into a preimmune fetus could be used in early treatment of many inherited hepatic metabolic diseases. This study was designed to assess the tolerance to hepatocyte transplantation and to test the feasability and toxicity of such an injection in a primate model. Fetal hepatocytes were obtained from two 120-day-old Macaca mulatta fetuses and cryopreserved. They were thawed, cultured in vitro, and transduced with a recombinant retrovirus expressing beta-galactosidase. Transduction efficiency was 75-85%. Three unrelated fetuses (90, 100, and 104 days old) were each given 1-2 x 10(7) transduced cells via the umbilical vein. This caused vasospasm and severe bradycardia. Two fetuses died in the 48 hours after transplantation; the third survived and was killed at the end of gestation. No evidence of the infused cells was found. Three fetuses (90 days old) were, therefore, given 3-4 10(7) hepatocytes by direct intrahepatic injection. All the fetuses survived without side effect. Donor cells were not apparent from histochemical staining and PCR reactions. There was no evidence of inflammatory reaction. These findings indicate that the protocole could be improved by increasing the number of transplanted cells and using specific hepatic promoters in the retroviral vectors to achieve an effective postnatal chimerism.

Preclinical studies for cell transplantation: isolation of primate fetal hepatocytes, their cryopreservation, and efficient retroviral transduction.

Fetal hepatocytes are an attractive target for in utero cellular transplantation. Their use could provide a very efficient way for implanting normal or transduced cells into the livers of affected fetuses. Marking cells with recombinant retroviruses is a powerful tool for evaluating the chimerism of grafted animals. The technique relies on the ex vivo transduction efficiency of the engrafted cells. We have isolated fetal primary hepatocytes from nonhuman primates. The cells were cultured and transduced with a retroviral vector carrying the Escherichia coli beta-galactosidase gene. Optimal gene transfer efficiency was obtained 48-60 hr after plating and was as high as 90%. Cryopreservation had little effect on cell viability and infectivity: The viability of thawed hepatocytes remained high (75-85%) and the infection efficiency was identical to that of freshly isolated cells. Efficient ex vivo retroviral gene transfer into fetal hepatocytes provides an appropriate system for testing allogenic grafting and for modifying immunogenicity of engrafted cells. These results open up new perspectives for cell transplantation through cell banking.

Hepatocyte growth factor expressed by a retrovirus-producing cell line enhances retroviral transduction of primary hepatocytes: implications for in vivo gene transfer.

Hepatocyte Growth Factor (HGF) is the more potent mitogen of mature hepatocytes. We have examined the effect of human HGF expression by a recombinant retroviral cell line (MFG-LacZ) on retroviral transduction of primary mouse and human hepatocytes. The HGF in the supernatant of MFG-LacZ cell line was correctly processed and biologically active. Transduction of mouse and human hepatocytes with the supernatant of transfected cells was increased 5-fold, as determined by beta-galactosidase activity. The production of HGF was stable and did not interfere with the viral titers of the producer cells. This study provides evidence that expression of HGF within a retrovirus-producer cell line increases the transduction rate of primary hepatocytes. Since the number of corrected cells is a limiting step for phenotypic correction of liver deficiencies, our approach should improve hepatic gene therapy efficiency. Furthermore this cell line should be useful for in vivo liver gene therapy.

[Towards gene therapy in familial hypercholesterolemia]. / Vers une thérapie génique de l'hypercholestérolémie familiale.

Familial hypercholesterolemia (FH) is an inherited disease caused by a defect in the gene encoding the Low Density Lipoprotein receptor (LDL-R). The ex vivo hepatic gene therapy which restore the expression of the normal protein in hepatocytes should correct the disease. Improved transduction efficiency and long lasting expression of the transduced gene remain the main goals of gene therapy research. We developed an efficient and reliable method for in vivo transduction of human, mouse and primate primary hepatocytes. A retroviral vector bearing the LDL-R cDNA driven by the liver-type pyruvate kinase promoter allows high and tissue specific expression of the gene in primary hepatocytes. A second vector with a housekeeping promoter corrects the LDL-R deficiency in fibroblasts from a FH patient. Ex vivo preclinical studies in non-human primates will provide new insight in transduced cells biology after reimplantation.

Activation of Moloney murine leukemia virus LTR enhances the titer of recombinant retrovirus in psi CRIP packaging cells.

The critical physiological function of the long terminal repeat in Moloney murine leukemia virus (MoMLV) gene expression has been thoroughly explored and shown to include binding sites for ubiquitous and tissue-specific transcription factors, such as the glucocorticoid responsive element (GRE) and the LVb sequence recognized by phorbol 12-myristate 13-acetate (TPA)-induced factors. The present study was undertaken to determine the effect of different activators, known to enhance expression of MoMLV, on their ability to modulate retroviral transcripts in psi CRIP producing cell lines. Improvement of recombinant retrovirus production by two psi CRIP producer cells was tested by using dexamethasone, TPA and sodium butyrate (Na-But) alone or in combination. We demonstrate that 5 mM Na-But, or 5 mM Na-But plus 1 microM dexamethasone significantly enhanced viral production. These compounds could induce a 10-fold increase in viral production. There was also a good correlation between the increased viral production and the titer measured after transduction of NIH 3T3. This improvement is of general interest, since a major goal for gene therapy is the production of high retroviral titer for increased transduction efficiency.

Familial isolated vitamin E deficiency. Extensive study of a large family with a 5-year therapeutic follow-up.

A major neurological deterioration, beginning with ataxia, led to the diagnosis of familial vitamin E deficiency in a girl. Based upon vitamin E determinations, 4/8 members of the (consanguineous) sibship were considered to be homozygous. Homozygosity was also found for the alleles of six markers linked to the AVED locus, recently identified in similar Tunisian or Sicilian families on chromosome 8q. Measures of vitamin E in lipoprotein fractions and in liver biopsy after vitamin E oral load suggested that free diffusion of vitamin E between the different compartments was possible and even increased. However, a high-affinity ligand seemed to be lacking, either in the hepatic recycling of vitamin E or in both the hepatic and the other vitamin E compartments. The 5-year substitutive treatment was successful only in the pre- or paucisymptomatic patients. Serum vitamin E must be measured in any unexplained progressive ataxia.

Two new polymorphisms in the coding sequence of the LDL receptor (LDLR) gene.

We identified by polymerase chain reaction/single-strand conformation polymorphism analysis two unreported polymorphisms in the low density lipoprotein receptor gene, located in exons 11 and 15. The exon 15 sequence variation can also be readily detected since it abolishes an MspI site.

Genetic exclusion of apo-B gene in recessive abetalipoproteinemia.

Abetalipoproteinemia is a recessive genetic disorder of unknown origin, which is characterized by absence of circulating apo-B-containing lipoproteins, malabsorption of intestinal fat, and degenerative neurological and retinal lesions. In this study, four families were analysed for genetic linkage between the abetalipoproteinemia phenotype and the apo-B genotype determined from polymorphisms of XbaI, MsPI, EcoRI and PvuII restriction sites and that of the 3'-minisatellite of the apo-B gene. The results definitively exclude mutation of the apo-B gene as a causal factor of abetalipoproteinemia in three families. Consanguinity of the parents in the fourth family made genotyping less conclusive.

Screening for new mutations in the LDL receptor gene in seven French familial hypercholesterolemia families by the single strand conformation polymorphism method.

To investigate the molecular basis of familial hypercholesterolemia (FH) in France, we applied the single strand conformation polymorphism (SSCP) method to the promoter region and the 18 exons of the low density lipoprotein receptor (LDLR) gene. Seven probands, 4 heterozygotes, 2 compound heterozygotes, and 1 homozygote, belonging to FH families were tested. In all cases, previous genetic analysis and/or LDL receptor fibroblast assay had shown that the disease was due to defects in the LDLR gene. Out of the nine mutations expected, one nonsense mutation in exon 2 and six missense mutations were identified in exons 3, 6, 8, 11, and 15. Two of the latter were found in exon 6. In each family, cosegregation of the base substitution and the disease was observed. Ninety-five control subjects were screened for the presence of the six missense mutations. None was detected, implying that the mutations identified are deleterious. Our results indicate that the SSCP analysis of amplified genomic DNA fragments can be successfully used to rapidly screen mutation containing exons in large genes. Furthermore, all these mutations are newly described and demonstrate heterogeneity of LDLR gene mutations responsible for FH in the French population, as in other reported Caucasian populations.

Recurrent mutation at aa 792 in the LDL receptor gene in a French patient.

Many mutations in the low density lipoprotein receptor gene (LDLR) have been characterized at the molecular level in individuals with familial hypercholesterolemia. Most of the mutations that have been reported are large deletions or insertions in the gene, it being much more difficult to identify point mutations. In this study, 139 unrelated French familial hypercholesterolemia subjects were screened for the presence of three different previously described LDLR point mutations, employing the polymerase chain reaction and allele-specific oligonucleotide hybridization. Only one subject carried a point mutation at amino acid position 792, which substituted a Trp codon for a Stop codon. The same mutation has previously been described in a subject originating from Saudi Arabia. Haplotype analysis of LDLR associated with each mutation was performed. The haplotypes were totally different, suggesting that this mutation has occurred more than once.

Extended haplotypes and linkage disequilibrium between 11 markers at the APOA1-C3-A4 gene cluster on chromosome 11.

One hundred fifty-four unrelated French Caucasian subjects were typed for 11 RFLPs at or near the APOA1-C3-A4 gene cluster on the long arm of chromosome 11. All subjects belonged to families having lived in the Toulouse area (in the southwest of France) for over three generations. Allele frequencies for each RFLP were in agreement with previous studies in Caucasian populations for the APOA1/SstI marker. Pairwise linkage disequilibrium was determined. Among the 55 pairs studied, 30 are newly reported. Only three significant nonrandom associations were found: APOA1/MspI-3'APOC3/SstI, APOA1/MspI-3'APOA4/XbaI, and APOA4/DraI-APOA4/XbaI. Extended 11-marker haplotypes were constructed. Haplotype frequencies were estimated by the maximum-likelihood procedure and compared to expected frequencies calculated under the assumption of equilibrium. Among the 37 estimated haplotypes, seven containing at least four nonrandomly associated alleles showed markedly increased frequencies. These results, obtained in a geographically homogeneous population, confirm the existence of disequilibrium in the apolipoprotein cluster, but to a lower extent than previously reported in Caucasian populations, which were geographically more heterogeneous.

Anderson's disease: genetic exclusion of the apolipoprotein-B gene in two families.

Anderson's disease is a recessive disorder characterized by intestinal fat malabsorption, absence of postprandial chylomicrons, and reduced levels of cholesterol, triglycerides, and apoproteins B, AI, and C. We have studied two families with, respectively, three and two children with Anderson's disease. Intestinal apo-B and apo-AIV mRNAs from two Anderson's patients were normal in size but their concentration was decreased fivefold compared with controls. After DNA digestion with seven restriction enzymes, restriction fragment length polymorphisms of apo-B gene did not show conclusive information except for Xba1, which revealed a lack of cosegregation between the restriction fragment length polymorphism and the Anderson's phenotype. Linkage analysis was performed using the polymorphism of the apo-B gene 3'minisatellite. Genomic DNA from parents and children was amplified by polymerase chain reaction using oligonucleotide primers flanking the apo-B gene 3'hypervariable locus. In both families each child inherited different apo-B alleles from at least one parent. According to the recessive mode of transmission of the disease, our results are incompatible with the involvement of the apo-B gene. More likely a posttranslational defect or a mutation in another gene encoding a protein essential for lipoprotein assembly or secretion may be involved.

A LDL receptor gene homozygous mutation: PCR amplification, direct genomic sequencing, associated haplotype, rapid screening for frequency.

Many mutations in the LDL receptor (LDLR) gene have now been identified mostly as gross gene rearrangements, however they only represent a weak percentage of all deleterious gene mutations causing Familial Hypercholesterolemia (FH). This discrepancy may be related to the difficulties in characterizing point or small defective mutations. In a three-generation family with Familial Hypercholesterolemia, one specific haplotype constructed with 12 intragenic restriction fragment length polymorphisms (RFLP) cosegregated with the disease, while in the consanguineous propositus there was homozygosity for this haplotype. By polymerase chain reaction (PCR) amplification followed by direct sequencing there was unequivocal evidence for a double dose of a unique mutation, (namely a duplication of 4 bases in exon 17), while there was a single dose in heterozygote relatives. We consequently screened a population selected under clinical and geographical criteria for this mutation by PCR and allele specific oligonucleotides (ASO) hybridization. None of the 158 type IIa individuals tested carried the same mutation. Herein, is a rapid combined genetic and molecular approach to characterize and evaluate the frequency of LDL Receptor gene mutations causing Familial Hypercholesterolemia, towards targeted prevention and therapy.