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  1. Science & Research (Biologics)

Toward More Effective Treatment of Blood Clotting Disorders: Pharmacogenomic Studies of ADAMTS13 and Related Proteins


Chava_Kimchi-Sarfaty headshot

Chava Kimchi-Sarfaty, Ph.D.

Office of Tissues and Advanced Therapies
Division or Plasma Protein Therapeutics
Hemostasis Branch



Dr. Kimchi-Sarfaty earned her M.Sc and Ph.D in Genetics from the Hebrew University and Hadassah Medical Center in Jerusalem. Her post-doctoral training was at the National Cancer Institute in Bethesda MD, working mostly on gene therapy. Subsequently, she moved to the FDA where she set up her independent research group in the Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies (OTAT) that focuses on pharmacogenetics and pharmacogenomics. Her research, which uses blood coagulation factors as model systems, is broadly applicable to therapeutic proteins, gene therapy, and gene editing. Her pioneering work that demonstrated that synonymous mutations affect protein folding and function overturned a dogma in biology and has wide ramifications in the basic understanding of biology, drug development, and the practice of medicine. Because synonymous mutations do not alter the amino acid sequence, they were assumed to be innocuous. She was among the first to demonstrate that this assumption is inaccurate.  Increased scientific awareness of the potential deleterious effects of synonymous mutations has led to the reevaluation of many diseases associated with so-called silent mutations. At the FDA, much of her research in synonymous mutations and codon optimization focuses on recombinant protein therapeutics and gene therapy, which often contain at least one synonymous mutation.  Her group has characterized many naturally occurring synonymous mutations with pathogenic implications, using both novel and existing protein characterization techniques and in silico tools.  In addition, based on her strong familiarity with coagulation factors, she reviews and chairs pre-INDs, INDs and BLAs for recombinant proteins and plasma derivatives products, such as von Willebrand factor, ADAMTS13, factor VIII, factor IX, thrombin, and fibrinogen.  Dr. Kimchi-Sarfaty works with the industry in support of the development of new coagulation recombinant proteins and she is a lead scientific reviewer of the Expert Committee on Biological Standardization applications.

General Overview

Our research group focuses on understanding the significance of the genetic sequences used to synthesize therapeutic proteins. Recombinant proteins (proteins made using genetic engineering) that are used to treat blood clotting disorders have several advantages over human plasma-derived products. Unlike the latter, which must be harvested from a pool of donor plasma, recombinant proteins can be manufactured cost-effectively under defined conditions, with decreased risk of viral contamination, increased product uniformity and flexibility in product design. Our research group focuses on understanding the significance of the genetic sequences used to synthesize these therapeutic proteins. Recently, we have expanded our research program to study how manipulation of viral genetic sequences can be used for vaccine development.

In all organisms, genetic sequences define the order in which amino acids are incorporated into a growing protein. These sequences have been shown to vary from person to person, sometimes without changing the amino acid backbone of a given protein. As a result, in recombinant protein production or gene therapy, industry has had flexibility to choose genetic sequences that are translated more efficiently and therefore are more cost effective. However, there is increasing awareness that variations in the sequence can significantly affect the protein's function, stability, distribution, and immunogenicity (how readily it triggers an immune response).  Furthermore, the expression system and the level of expression may also have such effects in recombinant proteins. To address this issue, we are developing sensitive laboratory assays that can explore subtle structural differences that may exist in therapeutic proteins.

Currently, we are using a combination of in silico (computer analysis) and laboratory-based assays to study the pharmacogenomics of three blood clotting proteins: factor IX (FIX), ADAMTS13, an anti-clotting factor, and its target, von Willebrand factor (VWF). FIX is an enzyme that helps carry out blood clotting and has been extensively studied over the past few decades. The absence of functional FIX within the blood leads to hemophilia B. FDA has approved both plasma-derived and recombinant FIX replacement products.  We are studying the role of ADAMTS13, the VWF-cleaving protease, in coagulopathy and thrombosis. We are examining test methods for the balance between VWF and ADAMTS13 as the cause of thrombosis in neonates and severe COVID-19 patients.

In order to engineer better and more effective recombinant proteins and gene therapy vectors, we have developed an online database that calculates the frequency with which different codons and codon-pairs occur in organisms, called Codon and Codon Pair Usage Tables (CoCoPUTs). Recently, we expanded CoCoPUTs to include transcriptomic data specific to different tissues (TissueCoCoPUTs). 

Since the outbreak of the COVID-19 pandemic, we were able to use these databases to design attenuated virus vaccines. Virus attenuation can be achieved through codon and/or codon pair deoptimization. By utilizing our expertise in codon/codon pair optimization we were able to promptly direct our efforts towards characterizing the virus sequence and developing deoptimization strategies. Collaborative efforts have been organized in order to validate experimentally these deoptimization strategies.

The in silico and in vitro methods that we are exploring may enable researchers and industry to produce safer therapeutic proteins and expediate vaccine development.  Currently, there are limited FDA guidelines regulating genetic sequences used to manufacture these therapeutics; the establishment of such guidance will help to ensure that only safe and effective therapeutics enter clinical development.

Scientific Overview

Chava_Kimchi-Sarfaty CoCoPUTs Screenshot

Today, there is unprecedented interest in gene engineering, for generation of recombinant therapeutic proteins, for gene therapy and for vaccine development.  Gene engineering design often includes non-synonymous and synonymous mutations. While amino acid substitutions are well recognized for their ability to alter protein structure and function, it was long assumed that synonymous mutations do not exert meaningful impact on expression, structure, functionality, or immunogenicity of the protein. However, there is increasing scientific evidence to the contrary.

FDA must develop guidelines for assessing the efficacy and safety of recombinant genes and the proteins they encode.  We are applying and constructing both computational and in vitro tools to better understand the consequences of synonymous mutations introduced by genetic engineering with an emphasis on codon and codon pair optimization or deoptimization.

The lack of scientific insight concerning how the genetic code impacts protein biogenesis represents a key regulatory hurdle.   We therefore seek to better describe these mechanisms experimentally using 1) naturally-occurring non-synonymous and synonymous mutations, 2) codon-optimized recombinant proteins, and 3) expression systems with multiple and single integration sites, to be able to assess the effect of expression levels on protein structure and function.  Currently, we are focusing on three therapeutic recombinant blood proteins:  ADAMTS13 (the von Willebrand factor (VWF)-cleaving protease), VWF and coagulation Factor IX (FIX). In addition, due to the COVID-19 pandemic, we expanded our studies to examine how synonymous codon substitutions in the form of codon pair deoptimization can lead to viral attenuation for vaccine development.

Our work has demonstrated that single synonymous mutations in FIX can induce mild hemophilia B through multiple cellular mechanisms. Additionally, our ongoing characterization of codon-optimized FIX has revealed variable changes to protein expression, structure and function. Through a novel molecular biology technique termed ribosome-profiling, we showed that codon optimization alters local translation kinetics.  Furthermore, we have demonstrated that several synonymous polymorphisms in ADAMTS13 can modulate its activity, conformation and stability and may rescue the phenotype when in conjunction with a deleterious ADAMTS13 mutation that would otherwise cause thrombotic thrombocytopenic purpura (TTP).

We are continuing to study genotype-phenotype relationships in FIX, ADAMTS13 and VWF, and we are expanding our studies to develop curated databases on several disease-associated genes with the aim to generate tools to predict disease based on genotype.
We have also developed a database with genomic codon and codon-pair usage data of species using the latest publicly available sequences. This will aide in genetic engineering techniques and broaden our understanding of genetic evolution.  We have expanded this tool to include tissue-specific codon usage to explore codon usage bias across human tissues.

An overarching goal of this work is to study gene expression, protein biogenesis, structure, and function.  This will facilitate development of standard protocols for assessing the safety and efficacy of recombinant therapeutic proteins. To this end, we have applied many established techniques to characterize the consequences of polymorphisms in FIX and ADAMTS13.  We are incorporating new techniques that harness NGS technology to more effectively assess in vivo mRNA structure, tRNA composition, and co-translational protein folding. 

Important Links


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