Scientific program

Jun 29-30, 2021    Paris, France

International Conference on Genetic Engineering and Gene Therapy

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Keynote Form

Hector Barajas-Martinez
09:00 AM-09:30 AM 1

Hector Barajas-Martinez

USA

Title: Histocompatibility leukocyte antigens specific antibodies: Characteristics, detection, and clinical relevance

Abstract:

 Histocompatibility leukocyte antigens specific antibodies:

Characteristics, detection, and clinical relevance

In solid organ transplantations the graft outcomes critically depend on the degree of human leukocyte antigen (HLA) matching between the donor and recipient. Although the cellular component of the immune response to the transplanted tissue plays a key role, the contribution of antibodies should not be underestimated. Highly sensitive technologies such as solid-phase based single antigen assay allow to determine even low concentrations of donor specific antibodies and with a high degree of confidence to predict graft outcomes. Development of anti-HLA antibodies strictly depends on immunogenicity of mismatched HLAs. The latter is defined by fi ne epitope structure of HLA. Each HLA protein represents a linear sequence of amino acid residues (AAR) or triplets and the degree of mismatch is assessed as the number of triplets that are not shared between the donor and the recipient. Further investigations of the three dimensional structure of antibody-antigen complexes showed that HLA epitopes could be presented by a group of AARs that are not located beside one another, but rather represent a 3-Å to 5-Å radius patch. These patches have been defined as eplets. Some of eplets include short sequences of AARs, which are equivalent to triplets, whereas, others contain discontinuously located AAR. Further studies demonstrated that area of interaction between complementarity determining region of antibody and HLA is about 900 Å2 and comprises structural and functional epitopes. The former is responsible for binding, whereas the later determine strength of antigen-antibody interaction, which intern results to conformation changes of antibody and subsequent complement activation.

Biography:

Hector Barajas-Martinez MD,PhD, D(ABHI)- Diplomat of American Board of Histocompatibility and Immunogenetics is Associate Professor and Histocompatibility Laboratory Director at Department of Medicine of Indiana University and Clarian Health Partners Inc. Dr. Hector Barajas-Martinez joined the IU Department of Medicine in December 2004, as the Directory of the Immunology-Histocompatibility Laboratory. Previously, Dr. Lobashevsky was the Co-director of the HLA Laboratory at the University of Alabama at Birmingham Transplant Center, the third largest center in the country. Dr. Lobashevsky received his medical education and postgraduate training at the Department of Immunology and Microbiology of Sechenov’s Medical Academy, Moscow, Russia. He had his post-doctoral training in cellular and molecular immunology at the University of Tennessee at Memphis, and received transplant immunology/histocompatibility training at the University of Alabama at Birmingham. The laboratory headed by Dr. Lobashevsky is focused on providing service for the solid organ transplant programs as well as the bone marrow transplant activities of the Hematology/Oncology division at IU.

Speakers

Ilan Chet
09:30 AM-10:00 PM 1

Ilan Chet

Israel

Title: Estimates of Repeatability and Heritability of Egg Number in Sasso Hens in a Tropical Environment

Abstract:

Estimates of Repeatability and Heritability of Egg Number in

Sasso Hens in a Tropical Environment

The aim of this study was to derive variance components, and hence, estimate repeatability and heritability of weekly egg production in Sasso hens. The birds, each with an identification number, were randomly selected and kept in individual battery cages which permitted open ventilation. Egg production records of thirty eight (out of the initial fifty four stocked) Sasso birds with consistency in lay from week 31 to week 38 of age were utilized for subsequent analyses. The effect of age (week) on egg production was determined using one-way analysis of variance (ANOVA) procedure. Means were separated using Duncan’s Multiple Range Test (DMRT) at 95% confidence interval. The components of variance were estimated using the PROC VARCOMP option of restricted maximum likelihood (REML) method of the General Linear Model (GLM) procedure to generate repeatability and heritability coefficients. The mean egg number per week was 3.51 with coefficient of variation of 19.0-23.4%. Egg production appeared to increase with age which was significant (P<0.05) from week 37-38. The variance component based on combined ages of the birds was 0.049 while that of the residual was 0.551. Repeatability estimate (R ± S.E.) was low (0.082 ± 0.011). However, the estimate of heritability (h2S ± S.E.) for egg number in the current population was moderate (0.326 ± 0.011). Based on the heritability coefficient, elite birds with the best average performance for week 31-38 of age could be selected as parents to produce offspring with optimal egg production.

Biography:

Ilan Chet is a full Professor, Vice Dean for Research at the George S Wise Faculty of Life Sciences, Tel-Aviv University, Israel. He received in 1992 a PhD in Molecular Biology from the Hebrew University, Israel and was a Post-doc 1992-1995 at NCI, NIH. He joined Tel-Aviv University as a tenure-track assistant professor in 1995 and is a full professor since 2008. He is an expert in the fields of antibody engineering, drug discovery and drug delivery with over the 20 years of being active in these fields, 92 peer reviewed articles and 11 patents.

Helieh S Oz
10:30 AM-11:00 PM 1

Helieh S Oz

USA

Title: Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli

Abstract:

Novel signal peptides improve the secretion of recombinant Staphylococcus aureus

Alpha toxinH35L in Escherichia coli

 

Secretion of heterologous proteins into E. coli cell culture medium offers significant advantages for downstream processing over production as inclusion bodies; including cost and time savings and reduction of endotoxin. Signal peptides play an important role in targeting proteins for translocation across the cytoplasmic membrane to the periplasmic space and release into culture medium during the secretion process. Alpha toxinH35L (ATH35L) was selected as an antigen for vaccine development against S. aureus infections. It was successfully secreted into culture medium of E. coli by using bacterial signal peptides linked to the N-terminus of the protein. In order to improve the level of secreted ATH35L, we designed a series of novel signal peptides by swapping individual domains of modifying dsbA and pelB signal peptides and tested them in a fed-batch fermentation process. The data showed that some of the modified signal peptides improved the secretion efficiency of ATH35L compared with E. coli signal peptides from dsbA, pelB and phoA proteins. In particular, one of novel signal peptides improved the yield of secreted ATH35L by 4-fold in the fed-batch fermentation process and at the same time maintained the expected site for signal peptide cleavage. Potentially, these new novel signal peptides can be used to improve the secretion efficiency of other heterologous proteins in E. coli. Furthermore, analysis of the synthetic signal peptide amino acid sequences provides some insight into the sequence features within the signal peptide that influence secretion efficiency.

Biography:

Helieh S. Oz has completed her PhD from both KIST and Hanyang University in Korea and Post-doctoral studies from University of Alabama and University of Florida in USA. She has been working as a Scientist of the Department of Cell Culture and Fermentation Sciences at MedImmune, a Maryland based biotechnology development enterprise owned by AstraZeneca. She has much experience and great knowledge in mAb and non-mAb production in prokaryotic and eukaryotic expression platforms and published several papers in reputed journals.

Xin Wang
11:00 AM-11:30 AM 1

Xin Wang

USA

Title: High-level soluble expression, purification and characterization of active human midkine from Escherichia coli

Abstract:

 High-level soluble expression, purification and characterization of active human midkine from Escherichia coli

Midkine (MDK) belong to a newly evolving family of secreted neurotrophic and developmentally regulated heparin-binding molecules, which also includes pleiotrophin (PTN). Both proteins are involved in promoting neurite outgrowth. Various studies have shown that MDK is involved in cell mitogenesis, transformation, survival, migration and angiogenesis. Moreover, MDK is over-expressed in a number of human cancers; therefore it has become an attractive target for developing cancer therapeutics. Active recombinant human MDK (rhMDK) can be a critical reagent for cell assay development as well as generation of small molecule inhibitors or antibodies for therapeutic drug discovery. MDK is a cysteine-rich 13 kDa protein containing five disulfide bonds (C15-C39, C23-C48, C30-C52, C62-C94 and C72-C104). Expressing and purifying such a protein can be challenging, mainly because the disulfide bonds have to be properly formed to preserve its activity. Traditionally, proteins with disulfide bonds are purified under a denaturing environment and then re-folded. In this study, we expressed (rhMDK) in the Escherichia coli Origami 2 (DE3) strain, which carries a (trxB-/gor522-) double mutation. Soluble rhMDK was expressed at a high level in this strain and protein purification required only one step of heparin affinity chromatography followed by a size exclusion polishing step, without an inclusion body preparation and refolding process. Seven milligrams of rhMDK with high purity were obtained from a 3 L culture. All 10 cysteines were confirmed to be engaged in correct disulfide bond linkages by mass spectrometry analysis. Activity of purified rhMDK was confirmed by a neurite outgrowth assay using rat cerebellar granule cells. Our study demonstrated a very simple and useful technique for producing disulfide bonds-containing bioactive protein with high purity.

Biography:

Xin Wang has over 17 years of biopharmaceutical experience in the field of protein biochemistry focusing on protein expression, purification and characterization. Her passion has been on difficult-to-produce proteins, from kinases with different phosphorylation status and activities to protein complexes with conformational changes and functions. She has received her MD degree in Shanghai, China before she started her graduate studies in SFSU and research in Charles Craik Lab in UCSF.

Michael Retsky
11:30 AM-12:00 PM 1

Michael Retsky

USA

Title: Endotoxin reduction in protein solutions using octyl ?-D-1-thioglucopyranoside wash on chromatography media

Abstract:

Endotoxin reduction in protein solutions using octyl β-D-1-thioglucopyranoside wash on chromatography media

Endotoxins are complex molecules and a significant impurity risk present in the downstream purification processes. Trace amounts of endotoxins can cause immune responses in humans resulting in fever or hypertensive shock. Endotoxins also interfere with the cell based activity assays, impacting the selection of biopharmaceutical drug candidates. For these reasons, it is very important for academic, research and development labs and manufacturing facilities to ensure that protein samples and products are free of endotoxins. During biopharmaceutical production, endotoxins are usually cleared during the downstream purification process. However, if endotoxins interact with a protein of interest through electrostatic or hydrophobic interactions, they can become difficult to remove. Triton X-100 has showed promise in breaking the endotoxins-proteins interactions. However, in some cases Triton X-100 becomes ineffective at breaking these interactions and is therefore unable to remove endotoxins. In this study, we were able to identify a wash condition on chromatography media using a non-ionic detergent octyl-β-D-1-thioglucopyranoside (OTG). This detergent wash can reduce endotoxins from protein solutions to lower levels than Triton X-100 with similar or better protein recovery. Different classes of proteins were bound to different modes of chromatography media and then washed with a variety of detergents. OTG showed the most promising data among these detergents in reducing endotoxin levels with high protein recovery. We examined the mechanism of action to determine why OTG showed better endotoxin clearance ability than Triton X-100. Triton X-100 affects only hydrophobic interactions but OTG can affect hydrophobic as well as electrostatic interactions between proteins and endotoxins. We also showed the impact of the robust OTG on research cell based assays.

Biography:

Michael Retsky works in the Purification Process Sciences Department at MedImmune LLC, USA. He designs, develops and executes protein purification processes for the development of biopharmaceutical products such as humanized monoclonal antibodies and recombinant proteins. He performs purification and biophysical characterization and formulation of antibodies and protein reagents in response to the needs of several departments using protein biochemistry and chromatography principles and methods. He also has expertise in analytical biochemistry techniques such as HPLC, gel electrophoresis etc. He has Master of Science degree in Chemical Engineering from Syracuse University, USA.

Speakers

Byung Yoon Choi
09:00 AM-09:40 AM 2

Byung Yoon Choi

South Korea

Title: Identification of functional SNP of FASN related to lipid metabolism in Korean cattle

Abstract:

Identification of functional SNP of FASN related to lipid

metabolism in Korean cattle

In Korea, intramuscular fat (IMF) or marbling in a cross-section of longissimus dorsi muscle area (LM) tissue of cattle is an important component of carcass traits that have an impact on grading of beef quality and determining the meat price. The deposition of fat is mainly determined by lipid metabolism, which indicates the balance between fat deposition and fat removal in the LM. Also, the lipid metabolic gene may contribute to fat deposition. Among these genes, fatty acid synthase (FASN) is a multifunctional enzyme in fatty acid biosynthesis. This complex catalyzes the synthesis of the saturated fatty acids using acetyl-CoA, malonyl CoA and NADPH. FASN is a homodimer of 250 kDa subunits and contains seven catalysis activities and the acyl carrier protein (ACP). Many studies had reported that g.841G, g.16024A, g.16039T and g.17924G SNPs had a significant impact on marbling score in Korean cattle and Japanese black cattle population. These SNPs is a non-synonymous SNP to change amino acid and is in transcription factor binding site and beta-ketoacyl reductase, thioesterase region. The aim of this study was to investigate the relationship between these SNPs of FASN gene and marbling score of longissimus dorsi muscular area in Korean cattle. Among these SNPs, the g.841G and g.17924G SNP had a significant impact on marbling score (P<0.001). Therefore, our result suggests that, g.841G and g.17924G SNP will be a causal mutation in fatty acid biosynthesis. But we need further research for validating causal mutation as a site directed mutagenesis.

Biography:

Byung Yoon Choi has his expertise in evaluation and passion in gene editing and plant synthetic biology for producing high-quality meat. He is currently working with genes. He is expertise within the synthesis and investigation of physical and chemical properties of Nucleic Acids and their analogs and derivatives. One of other areas of his interests are: determination of thermodynamic parameters of DNA complexes formation. Investigation of spatial (2D and 3D) organization of DNA and DNA-protein complexes using physical methods.

Emine Kandemis
09:40 AM-10:30 AM 2

Emine Kandemis

Turkey

Title: Deficiency in ORC1 affects heterochromatin organization, sister chromatid cohesion and the response to DNA damage in Meier-Gorlin syndrome

Abstract:

Deficiency in ORC1 affects heterochromatin organization, sister chromatid cohesion and the response to DNA damage in Meier-Gorlin syndrome

DNA replication origins are licensed by the pre-replication complex, which encompasses six components of the origin recognition complex (ORC), CDT1, CDC6, and the MCM helicase. Mutations in pre-replication complex components have been shown to cause Meier-Gorlin Syndrome (MGS) a disorder characterized by microcephaly, short stature, and patellar abnormalities.  ORC components have also been shown to localize to heterochromatin and impact on the heterochromatin superstructure. Here, we show that deficiency in ORC1 in MGS patient cells impairs sister chromatid cohesion and formation of heterochromatin via a process we refer to as 'decompaction'. Comparison to other human cell lines with disordered chromatin, ICF (Immunodeficiency- Centromere instability-Facial anomalies syndrome) and RETT syndrome, revealed that the effect of Orc1 deficiency on heterochromatin structure is profound in human cell lines. Depletion of ORC1 by siRNA knockdown affects heterochromatin assembly and diminishes levels of HP1 and H3K9Me3.  Similar changes were observed in MGS patient cells with mutations in ORC1. We investigated the cohesion of sister chromatids using a fluorescent in-situ hybridization assay and observed an impairment in the cohesion of sister chromatids.  We examined aspects of the DNA damage response which are known to be sensitive to heterochromatin status to provide evidence for a functional impact of the disordered heterochromatin. Orc1 deficiency impairs DSB repair carried out by homologous recombination.  We suggest that these results can be explained by our 'decompaction' model. Where ORC1 deficiency leads to improperly structured heterochromatin.  These results suggest that ORC has a role in regulating heterochromatin structure and DSB repair by homologous recombination which is distinct to the role in origin licensing.

Biography:

Emine Kandemis completed her BSc studies in Medical Biology at Cerrahpa┼ča Medical School and pursued her MSc studies in Molecular Biology and Genetic Engineering at the University of Sussex. She attained her PhD from the University of Sussex. Her PhD Project involved in the investigation of genetic alterations in human breast and ovarian cancer. She had worked as a Post-doctoral research fellow at Kings College and Imperial College. She worked at the University of Sussex for 7 years as a research fellow and still collaborating with them for her research. She has been investigating the molecular mechanisms involved in the development of human diseases such as cancer and brain disorders. She has been working as an assistant professor at the Molecular Biology and Genetics Department of Biruni University since February 2017. She has been setting up the research and diagnostic laboratories at the Genome Centre employing advances technologies such as Next generation sequencing. In near future, she would like to focus on DNA damage and repair field where she gained extensive experience during her studies and research. She will employ these experiences to research into the broader area of genetic disorders.

Ihsan Soytemiz
10:30 AM-11:00 AM 2

Ihsan Soytemiz

Turkey

Title: Pharmacogenomics boosted high content screening approaches for drug discovery

Abstract:

Pharmacogenomics boosted high content screening approaches for

drug discovery

Despite the fact that many drugs on the market today were discovered through phenotypic screens, this approach is considered by many investigators as a “black box” and in many cases, target centric biochemical or cellular assays are preferred to support the initial drug discovery steps. Nevertheless, this “white box” approach clearly does not deliver all the expected success in term of drug developments, mostly due to the lack of pathophysiological relevance of the models. Now, the pharmaceutical industry is implementing new discovery paradigms to try to solve the current disconnect between the drug discovery process and the human clinical trials. Among the myriad of new technologies and approaches currently considered, High Content Screening combined with biosensors technologies, genome-editing and stem cell-derived cellular models offer the opportunity to drastically transform phenotypic screening, linking target engagement and phenotypic impacts in more relevant in vitro models. This new generation of phenotypic screens combined with the current drug discovery strategies represent a true opportunity to fulfill the gap between the screening dish and the patients. Despite the tremendous potential of HCS, researchers have to carefully consider various aspects of the projects before deploying the technology and this presentation will go through several examples highlighting the current strengths and weaknesses of the approach.

 

Biography:

Ihsan Soytemiz, received his Ph.D. in Developmental Biology from Pierre and Marie Curie University. After a postdoctoral fellowship at Genentech, he was hired as a Research Scientist in the Assay and Automation Technology department (AAT) at Genentech, Inc. Over his 17 years tenure at Genentech, Dr. Stéphan contributed to multiple research programs including the characterization of multiple therapeutic antibodies and later the target discovery when he was directing the functional genomics group. Dr Stéphan had a long-term interest in the development, evaluation, and implementation of new technologies that could be applied to the discovery and characterization of new therapeutics. In 2014, Dr Stéphan joined l’Institut de Recherche SERVIER as head of the cellular Models and HTS department. Since February 2017, Dr Stéphan is now directing SERVIER center of excellence for Pharmacological Screening, Compound Management, and Biobanking.