ESHG Posters 23

C. Hesling¹, M. D'Incan², C. D'Incan¹, P. Souteyrand², Y. J. Bignon¹;
¹Laboratoire d'Oncologie Moléculaire, Centre Jean Perrin, Clermont-Ferrand, FRANCE, ²Hôtel-Dieu CHRU, Clermont-Ferrand, FRANCE.

Background: Melanoma tumors are known to be radioresistant. Recent studies showed that BRCA1 (BReast CAncer 1) is part of a multiprotein complex called BASC, involved in recognition and repair of aberrant DNA structures such as the double strands breaks induced by X-ray irradiation.
Aim of the study: to determine the role of BRCA1 expression in the radioresistance of melanoma cell lines.
Materials and Methods: The A375 melanoma cell line was transfected with a vector expressing an anti-BRCA1 ribozyme. Clones with diminished mRNA expression determined by real time quantitative RTPCR were irradiated and cell survival assays performed.
Results: Two clones, c88 and c91, exhibited a residual BRCA1 expression of 60% and 50% respectively. Both had increased radiosensitivity compared to non transfected lines and lines transfected with the vector alone. Clone 88 was cultivated for two months, after which it recovered a normal BRCA1 level of expression and interestingly also its radioresistance.
Discussion: Our data suggest that in melanoma as it has been shown in BRCA1 mutated breast cell lines, BRCA1 expression is involved in radioresistance. Work is in progress to study the pathways impaired after BRCA1 down regulation using a multigenetic approach with DNA chips.

N. Huzjak¹, I. Barisic¹, A. Resic¹, V. Kusec², D. Anticevic³, D. Dodig², D. Primorac⁴;
¹Childrens University Hospital Zagreb, Zagreb, CROATIA, ²Clinical Hospital Centre Rebro, Zagreb, CROATIA, ³Clinical Hospital Centre Salata, Zagreb, CROATIA, ⁴Clinical Hospital Centre Split, Split, CROATIA.

Background: Severe osteogenesis imperfecta (OI) is a hereditary disorder characterised by increased bone fragility and progressive bone deformity. So far, no effective medical treatment is available. As secondary osteoporosis is an important feature of OI, antiresorptive activity of the aminobisphosphonates may improve clinical outcome in children.
Aim: to assess the clinical impact of the administration of bisphosphonates in Croatian OI patients.
Methods: We report results of 1-3 years treatment with intravenous pamidronate (APD) in seven children (four girls) of age 3 months - 11 years at entry, with severe OI. Pamidronate was administered in cycles as monthly infusions at a daily dose of 1-1,5 mg/kg during 6 months following pause for three months, or the same dose for three days every four months.
Results: Following treatment DEXA measurements showed a gradual increase in bone density in all patients. Number of confirmed fractures decreased in all. The reduction in pain and improvement in well-being and ability were impressive in two boys who had been confined to a wheelchair and now they walk using crutches. Acute phase reactions were noted during first infusion cycle in two children and asymptomatic hypocalcemia in three children. Three children gained excessive weight.
Conclusion: although bisphosphonates do not correct basic abnormalities in OI, they significantly alter the natural course of the disease and improve patients’ quality of life. For the time being they seem not only effective but also devoid of any adverse effects on bone growth and remodelling.

Liposuction - a less invasive surgical method of debulking plexiform neurofibromas

D. Babovic-Vuksanovic¹, U. Bite¹, S. Babovic²;
¹Mayo Clinic, Rochester, MN, ²Olmsted Medical Center, Rochester, MN.

Neurofibromatosis type I (NF1) is a common autosomal dominant disorder in humans. The hallmark of NF1 is development of neural tumors. Plexiform neurofibromas are a major source of morbidity associated with NF1. These tumors are often large and inconveniently located, leading to disfigurement and compromise of vital structures by compression and tissue infiltration. Surgery still remains the only therapeutic option for patients on whom a tumor is causing disability or pain. In many patients tumors are not completely resectable and there is a high rate of tumor re-growth. Surgical removal of tumors is associated with high risk of damage of surrounding vital structures as well as with significant hemorrhage. Also, surgical debulking of tumors sometimes leads to extensive scaring which may be very disfiguring. We report a novel approach in surgical therapy of plexiform neurofibromas using liposuction in two patients. This method is less invasive than a conventional surgical tumor debulking. The procedure is well tolerated and post surgical recovery is short. Liposuction may be a prefered surgical method for debulking of superficial plexiform neurofibromas in patients with NF1.

E. Bonifazi¹, F. Sangiuolo¹, M. Lais¹, G. Maira², A. Mangiola², G. Novelli¹;
¹Tor Vergata University, Department of Biopathology, Human Molecular Genetics Unit, Rome, ITALY, ²Institute of Neurosurgery, Catholic University, Rome, ITALY.

Vascular endothelial growth factor (VEGF) is a positive effector of angiogenesis, encouraging neovascularization and growth of cancerous cells.We used an innovative gene modification approach based on Small Fragment Homologous Replacement (SFHR) technique for down-regulation of VEGF expression in human glioblastoma cell lines (U87). SFHR allows the modification of specific genes in situ by a gene targeting approach. The method is based on the introduction of small fragments of DNA into cells, that pair and replace the homologous endogenous sequence with the introduced fragment. We transfected U87 cells with a mutagenized DNA fragment homologous to exon 3 sequence of the VEGF gene. The corrective fragment was complexed with cationic liposome (Gene Porter, GTS) at different liposome to DNA charge ratio (+/-).The fragment was designed to insert a stop mutation in the wild type sequence in order to create a truncated and unstable VEGF protein. In addition, a unique Dde I restriction site was also inserted as a positive marker for replacement. Recombination at the appropriate genomic locus and expression of the modified CFTR mRNA were assayed using PCR amplification and restriction analysis. ELISA test was also performed to quantify residual VEGF protein secreted by cells. Molecular DNA analysis shows recombinant restriction pattern (DdeI) for lipid/charge ratio (+/-) from 42/1 to 2.7/1.The recombinant allele was absent at neutral charge of the complex.This study extends the efficacy of SFHR as a gene targeting techniques to introduce gene modifications able to inhibit expression of selected genes.
Work supported by Ministero dell'Istruzione e Ricerca Scientifica

Persistent failure of RNA/DNA oligonucleotides (chimeraplasts) in gene correction : targeting the HPRT gene

J. Albuquerque-Silva¹, G. Vassart¹^,2, J. Lavinha³, M. J. Abramowicz²^,1;
¹Laboratory of Medical Genetics-ULB, Brussels, BELGIUM, ²University Hospital Erasme-ULB, Brussels, BELGIUM, ³Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisbon, PORTUGAL.

Initial studies of gene correction using chimeric RNA/DNA oligonucleotides (RDOs), also named chimeraplasts, reported exciting rates of correction of point mutations (up to 40% in one in vivo model). However, the technique has not yet held its promises in the fields of transgenics and gene therapy, and some groups reported persistent failures.
In order to implement the technique in our laboratory, we targeted the HPRT gene. HPRT+ and HPRT- cells can be readily selected in HAT and in 6-thioguanine (6-TG) medium, respectively. The gene is expressed ubiquitously and is located on the X chromosome so that only one allele is to be mutated in male (XY) cells. We aimed at introducing a previously reported human mutation, which leaves virtually no residual enzymatic activity in functional assays. We introduced mutated and control RDOs in human male, diploid cell lines by transfection or direct microinjection. In all experiments we failed to observe any significant difference in the number of 6-TG-resistant clones between controls and RDO-treated cells. Moreover, resistant clones did not result from specific mutagenesis but merely from the background noise of the selection process.
Our experiments were designed to meet criteria defined to address criticisms expressed about early reports dealing with RDOs. In view of potential artifacts and the lack of reproducibility of published reports, we consider that conversion mediated by chimeric RDOs still awaits validation. We hope that confrontation of negative as well as positive results will help solving the problem of reproducibility of this potentially promising methodology.

A. Laner¹, A. Ramalho², S. Cattani¹, S. Christan¹, B. Marques², S. Beck², M. Amaral²^,3, D. Schindlbauer⁴^,1;
¹Dept. Medical Genetics, Childrens Hospital, Ludwig Maximilians University, Munich, GERMANY, ²Centre of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisbon, PORTUGAL, ³Faculty of Sciences, University of Lisbon, Lisbon, PORTUGAL, ⁴Institute of Human Genetics, Technical University, Munich, GERMANY.

We engineered and initially analyzed expression of a 145 kb fusion gene. Starting from a genomic P1 based artificial chromosome (PAC) clone of the cystic fibrosis conductance regulator gene (CFTR) and the enhanced green fluorescence protein (EGFP) cDNA, a seven step cloning procedure resulted in a genomic CFTR-EGFP fusion construct (CG2). It contains approximately one half of the genomic CFTR gene locus from position -60 kb to exon 10, the EGFP cDNA replacing CFTR exons 10-24, and 2 kb of 3' sequences of the CFTR gene. Lipofection of the human lung sarcoma cell line HT1080 resulted in integration of one or possibly few copies of the construct into a host chromosome. Expression and correct splicing of the synthetic 10 exon gene has been confirmed by RT-PCR. Since expression of the primary transcript of CG2 requires intact transfer of a minimum of 77 kb, the reporter should be useful for the development of large DNA transfer protocols. The reporter within it's own chromatin context now allows the analysis of transgene expression within target tissues

An engineered genomic CFTR-GFP fusion gene is expressed and correctly spliced on human artificial chromosomes.

D. Schindelhauer¹^,2, A. Laner², S. Christan², S. Cattani², A. Ramalho³, B. Marques³, S. Beck³, M. Amaral³^,4;
¹Institute of Human Genetics, Technical University, Munich, GERMANY, ²Medical Genetics, Ludwig Maximilians University, Munich, GERMANY, ³Centre of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisboa, PORTUGAL, ⁴Faculty of Sciences, University of Lisboa, Lisboa, PORTUGAL.

To assess co-lipofection of telomerized components cloned into a ditelomeric P1 phage based artificial chromosome vector (pTAT) as a means to incorporate genomic genes into human artificial chromosomes (HAC), we use our recently engineered, telomerized CFTR-EGFP fusion gene construct CGT21 (138 kb) which expresses a 77kb primary transcript under the control of the CFTR promoter. Comparably small amounts (100 ng) of CGT21 and telomerized alpha satellite DNA of chromosome 17 (a17T, 200 kb) where sufficient to generate a large number of co-transfected, blasticidin S resistant clones. Presence of the centromere efficiently changed the fate of the transferred gene from integration to HAC formation in 5 out of 5 lines analyzed, out of which 3 expressed and correctly spliced all 10 exons. Low copy HACs were detected by FISH in only 10-50% of metaphases, regardless whether on or off selection. Nevertheless, the majority (>87%) of single cell derived subclones which have been isolated after having been off selection for 30 generations, grew upon re-selection, indicating stable HACs in virtually all cells. While two of the lines presented stable HACs without integration after 60 generations, one HAC line showed an increasing proportion of metaphases (up to 40%) with integrations in two independent (painting probe) host chromosomes, indicating secondary events in this HT1080 cell clone. The data suggest that de novo HAC formation from naked DNA is an efficient means to stably transfer genomic copies of genes which can be engineered ad libitum in PACs.

Safety and efficacy of recombinant acid alpha-glucosidase (rhGAA) in patients with classical infantile Pompe disease

L. Klinge, V. Straub, U. Neudorf, K. Goerlinger, T. Voit;
Children's Hospital, University of Essen, Essen, GERMANY.

Purpose: Pompe disease is an autosomal recessive muscle-wasting disorder caused by deficiency of the lysosomal enzyme GAA. Classical infantile Pompe disease is characterized by progressive cardiomyopathy, muscle weakness, respiratory insufficiency and death in early infancy. Methods: Evaluated are data of four patients with classical infantile Pompe disease participating in two separate Phase 2 open-label, multinational multicenter studies. Two patients are treated with rhGAA derived from milk of transgenic rabbits 40 mg/kg IV weekly and two patients with CHO-cell derived rhGAA 10 mg/kg IV weekly. Safety is evaluated by recording adverse events, vital signs, physical examination, antibodies to rhGAA and routine clinical lab tests. Clinical efficacy endpoints include ventilator-free survival, left ventricular mass, motor and cognitive development, and growth. Muscle biopsies are performed at baseline, 3 months, and 12 months. Two patients have been treated for more than one year and two for three months by now (mean age at enrollment 6,6 months, range 2,6-14,5). Results: Initial safety data indicates that the treatment is generally well-tolerated. There has been an overall improvement in left ventricular mass, cardiac function, skeletal muscle function and histologic appearance as evidenced by reduction of muscle glycogen. Younger patients and those with less advanced disease have shown the best clinical response. Conclusions: Our data suggest that rhGAA appears to be well-tolerated and capable of improving cardiac status and skeletal muscle function in patients with classical infantile Pompe disease. Further long-term safety and efficacy data are required to assess the potenial of this therapy.