651. Oligonucleotide-Mediated Gene Repair Restores Full Length SMN mRNA Expression in Mutant-SMN Murine Fibroblasts

Spinal Muscular Atrophy (SMA) is a severe neuromuscular disease characterized by degeneration of a-motor neurons in the spinal cord. Ninety percent of patients affected by SMA have deletion of the Survival of Motor Neuron-1 (SMN1) but they retain a copy of the gene (SMN2) in their genome. SMN2 produces almost no functional SMN protein due to a CAET transition in exon 7 that disrupts a splicing enhancer sequence and causes skipping of exon 7 in >90% of the processed SMN mRNA. As a consequence, SMA cells have a decreased amount of properly spliced full length SMN mRNA, which encodes functional SMN protein. This decrease in functional SMN protein leads to decreased survival of motor neurons. Many attempts have been made to increase functional SMN protein levels from the SMN2 gene by correcting the splicing-process defect

In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique

BACKGROUND: SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex. METHODS: In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and ΔF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM). RESULTS: We demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio. CONCLUSIONS: These studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization

Periodontal condition in growing subjects with Marfan Syndrome: a case-control study

Background Marfan’s syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in the extracellular matrix protein fibrillin-1. Orofacial characteristics may be useful in identification of the syndrome. Severe periodontitis is sometimes observed in MFS patients, but no in-depth information has been reported in Italian groups of growing subjects with MFS. The aim of this study was to analyze the periodontal condition on a group of growing subjects affected by MFS, in comparison with a typically developed control group. Methods A group of 16 subjects with diagnosed MFS were recruited from the Centre for Rare Diseases for Marfan Syndrome and Related Disorders of Tor Vergata University Hospital. The Marfan Group (MG) was compared with a Control Group (CG) composed by 20 nonsyndromic subjects. The periodontal clinical parameters like Marginal Gingival Thickness (GT), Plaque Index (PI), Bleeding On Probing (BOP) and Modified Periodontal Screening and Recording (PSR) were assessed. Results The mean value of PI in MG was 59%, instead in CG it reached 21%. Analysis showed a significant difference between MG and CG also for the BOP. In MG the mean value of BOP attained 36% and in CG it reached 16%. A statistical significant difference of distribution of PSR index between the two groups was found for all sextant examined. Discussion Patients with Marfan syndrome reveal a higher presence of plaque and consequently a generalized inflammation in the oral cavity when compared with a control group

Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids

The global health emergency for SARS-CoV-2 (COVID-19) created an urgent need to develop new treatments and therapeutic drugs. In this study, we tested, for the first time on human cells, a new tetravalent neutralizing antibody (15033-7) targeting Spike protein and a synthetic peptide homologous to dipeptidyl peptidase-4 (DPP4) receptor on host cells. Both could represent powerful immunotherapeutic candidates for COVID-19 treatment. The infection begins in the proximal airways, namely the alveolar type 2 (AT2) cells of the distal lung, which express both ACE2 and DPP4 receptors. Thus, to evaluate the efficacy of both approaches, we developed three-dimensional (3D) complex lung organoid structures (hLORGs) derived from human-induced pluripotent stem cells (iPSCs) and resembling the in vivo organ. Afterward, hLORGs were infected by different SARS-CoV-2 S pseudovirus variants and treated by the Ab15033-7 or DPP4 peptide. Using both approaches, we observed a significant reduction of viral entry and a modulation of the expression of genes implicated in innate immunity and inflammatory response. These data demonstrate the efficacy of such approaches in strongly reducing the infection efficiency in vitro and, importantly, provide proof-of-principle evidence that hiPSC-derived hLORGs represent an ideal in vitro system for testing both therapeutic and preventive modalities against COVID-19

Comparative analysis between saliva and buccal swabs as source of DNA: lesson from HLA-B*57:01 testing

Aim: Our work aimed to designate the optimal DNA source for pharmacogenetic assays, such as the screening for HLA-B*57:01 allele. Materials & methods: A saliva and four buccal swab samples were taken from 104 patients. All the samples were stored at different time and temperature conditions and then genotyped for the HLA-B*57:01 allele by SSP-PCR and classical/capillary electrophoresis. Results: The genotyping analysis reported different performance rates depending on the storage conditions of the samples. Given our results, the buccal swab demonstrated to be more resistant and stable in time with respect to the saliva. Conclusion: Our investigation designates the buccal swab as the optimal DNA source for pharmacogenetic assays in terms of resistance, low infectivity, low-invasiveness and easy sampling, and safe transport in centralized medical centers providing specialized pharmacogenetic tests

A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG)v(TCTG)w(CCTG)x(NCTG)y(CCTG)z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG)n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling

Novel mutations of TCOF1 gene in European patients with treacher Collins syndrome

Background: Treacher Collins syndrome (TCS) is one of the most severe autosomal dominant congenital disorders of craniofacial development and shows variable phenotypic expression. TCS is extremely rare, occurring with an incidence of 1 in 50.000 live births. The TCS distinguishing characteristics are represented by down slanting palpebral fissures, coloboma of the eyelid, micrognathia, microtia and other deformity of the ears, hypoplastic zygomatic arches, and macrostomia. Conductive hearing loss and cleft palate are often present. TCS results from mutations in the TCOF1 gene located on chromosome 5, which encodes a serine/alanine-rich nucleolar phosphoprotein called Treacle. However, alterations in the TCOF1 gene have been implicated in only 81-93% of TCS cases. Methods: In this study, the entire coding regions of the TCOF1 gene, including newly described exons 6A and 16A, were sequenced in 46 unrelated subjects suspected of TCS clinical indication. Results: Fifteen mutations were reported, including twelve novel and three already described in 14 sporadic patients and in 3 familial cases. Moreover, seven novel polymorphisms were also described. Most of the mutations characterised were microdeletions spanning one or more nucleotides, in addition to an insertion of one nucleotide in exon 18 and a stop mutation. The deletions and the insertion described cause a premature termination of translation, resulting in a truncated protein. Conclusion: This study confirms that almost all the TCOF1 pathogenic mutations fall in the coding region and lead to an aberrant protein