Vaccines, Blood & Biologics

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

Principal Investigator: Chava Kimchi-Sarfaty, PhD
Office / Division / Lab: OTAT / DPPT / HB

General Overview

Recombinant proteins (proteins made using genetic engineering) are used to treat blood clotting disorders and 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.

In all organisms, genetic sequences define the composition of unique proteins by designating the exact amino acids to be 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, industry has had flexibility to choose genetic sequences that are cost and time 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). Currently, there are limited FDA guidelines that govern the genetic sequences used to manufacture these therapeutics; the establishment of such guidance will better ensure that only safe and effective therapeutics enter clinical development.

Our research program strives to elucidate the true impact of genetic variation on recombinant protein properties (pharmacogenomics). This work will provide the FDA with the scientific evidence on which to base guidelines and regulation for the production of protein-based therapeutics. Currently, we are studying the pharmacogenomics of two blood clotting proteins, ADAMTS13 and factor IX (FIX), using a combination of in silico (computer analysis) and laboratory-based assays. ADAMTS13 is an anti-clotting factor whose gene was only recently discovered. Factor IX (FIX), which helps carry out blood clotting, has been extensively studied over the past few decades. The absence of functional FIX within the blood leads to hemophilia B. The Agency has approved both plasma-derived and recombinant FIX replacement products.

In order to study these clotting factors, we are working to establish computer-based systems that can compare genetic variations and predict their impact on protein properties. We are simultaneously developing sensitive in vitro assays that can be used to highlight subtle structural differences that may exist in therapeutic proteins produced using modified DNA sequences. Both of these in silico and in vitro methods will help researchers and industry determine the expression, function, and conformation (shape) of ADAMTS13, FIX, and eventually, other clotting factors.

This work involves the analysis of genetic changes that are (1) intentionally engineered into a protein expression sequence and (2) gene variants that naturally exist in the human population, where genotype-phenotype connections can be drawn from the laboratory to clinical outcome (e.g. bleeding, thrombosis). The overarching goal of our work is to improve our understanding of blood clotting proteins and to support the development of appropriate regulatory oversight of these products, which will ultimately ensure their safe manufacture and effective use.

Scientific Overview

The number of therapeutic applications for recombinant proteins continues to expand. The first recombinant replacement clotting factor was licensed over two decades ago. Today, these molecules are subjects of cutting-edge protein design, which increasingly includes engineering synonymous or non-synonymous mutations into the expression sequence to improve protein expression and production. It was long assumed that synonymous (silent) mutations in genes cannot affect the expression, functionality, half-life or immunogenicity of the proteins they encode. Similarly, it is frequently believed that a therapeutic protein harboring common, single nucleotide polymorphisms can be used to treat all types of patients. However, there is increasing scientific evidence to the contrary. Thus, the FDA is at a critical juncture as it becomes clear that guidelines must be developed for assessing the efficacy and safety of recombinant proteins, including the sequences that encode them.

Our investigation centers on splicing forms, synonymous mutations and various polymorphisms in therapeutically relevant proteins, with a particular focus on ADAMTS13 (the von Willebrand factor-cleaving protease) and coagulation Factor IX (FIX). At present, our results demonstrate that a single synonymous mutation in FIX is capable of causing hemophilia B through multiple cellular mechanisms. We are now using bioengineered recombinant FIX proteins to understand the consequences of introducing multiple synonymous mutations in the same expression sequence to engender increased protein production, a process termed codon-optimization. We are interested in assessing the protein properties of codon-optimized proteins and the method (algorithm) used to create them.

Our work on ADAMTS13 has shown that several synonymous polymorphisms in ADAMTS13 change the specific activity, conformation and stability of this molecule. We are now studying genotype-phenotype relationships in unique patient populations (e.g. congenital heart disease) to better understand and identify clinical consequences of genetic variants in ADAMTS13. Findings such as these can aid in the diagnosis and clinical management of patients. Importantly, the testing methods we use to monitor gene expression, protein biogenesis, protein structure and function will facilitate the development of standard testing protocols for validating the safety and efficacy of therapeutic proteins. We anticipate that developing in silico criteria for narrowing sequencing options will reduce the cost and time required to develop recombinant proteins and improve their quality.

We have assessed the following methods to monitor the consequences of single mutations and polymorphisms in FIX and ADAMTS13: (1) mRNA- qRT-PCR; (2) intracellular expression- flow cytometry and Western blotting; (3) protein structure/conformation- comparing conformation-sensitive antibody affinity using reducing and non-reducing PAGE/Western blot, determining kinetics and affinities of protein-antibody interaction using biosensors, analyzing trypsin digestion patterns, circular dichroism; dynamic light scattering; (4) protein secretion- assessment in the presence or absence of proteasome and lysosome inhibitors; (5) extracellular function- analyzed with multiple activity assays that assess cleavage of synthetic and native substrates. We are also actively incorporating new techniques (e.g. ribosome profiling) that harness next generation sequencing technology to more pointedly assess the kinetics of protein translation, which can ultimately lead to altered co-translational protein folding.



Thromb Res 2017 Oct;158:98-101
Compounding variants rescue the effect of a deleterious ADAMTS13 mutation in a child with severe congenital heart disease.
Katneni UK, Hunt R, Hettiarachchi GK, Hamasaki-Katagiri N, Kimchi-Sarfaty C, Ibla JC

BMC Bioinformatics 2017 Sep 2;18(1):391
A new and updated resource for codon usage tables.
Athey J, Alexaki A, Osipova E, Rostovtsev A, Santana-Quintero LV, Katneni U, Simonyan V, Kimchi-Sarfaty C

J Med Genet 2017 May;54(5):338-45
Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B.
Simhadri VL, Hamasaki-Katagiri N, Lin BC, Hunt R, Jha S, Tseng SC, Wu A, Bentley AA, Zichel R, Lu Q, Zhu L, Freedberg DI, Monroe DM, Sauna ZE, Peters R, Komar AA, Kimchi-Sarfaty C

Blood 2017 Feb 16;129(7):896-905
A mechanistic investigation of thrombotic microangiopathy associated with intravenous abuse of Opana ER.
Hunt R, Yalamanoglu A, Tumlin J, Schiller T, Baek JH, Wu A, Fogo AB, Yang H, Wong E, Miller P, Buehler PW, Kimchi-Sarfaty C

F1000Res 2017 Feb 7;6:113
Recent advances in (therapeutic protein) drug development.
Lagasse HA, Alexaki A, Simhadri VL, Katagiri NH, Jankowski W, Sauna ZE, Kimchi-Sarfaty C

Haemophilia 2017 Jan;23(1):e8-17
The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia.
Hamasaki-Katagiri N, Lin BC, Simon J, Hunt RC, Schiller T, Russek-Cohen E, Komar AA, Bar H, Kimchi-Sarfaty C

Pers Med 2015;12(4):403-15
Personalized approaches to the treatment of hemophilia A and B.
Simhadri VL, Banerjee AS, Simon J, Kimchi-Sarfaty C, Sauna ZE

PLoS One 2015 Jul 15;10(7):e0132433
Small ncRNA expression-profiling of blood from hemophilia A patients identifies miR-1246 as a potential regulator of Ffctor 8 gene.
Sarachana T, Dahiya N, Simhadri VL, Pandey GS, Saini S, Guelcher C, Guerrera MF, Kimchi-Sarfaty C, Sauna ZE, Atreya CD

Trends In Genetics 2014 Jul;30(7):308-21
Exposing synonymous mutations.
Hunt RC, Simhadri VL, Iandoli M, Sauna ZE, Kimchi-Sarfaty C

Br J Haematol 2014 Apr;165(1):154-8
Single-nucleotide variations defining previously unreported ADAMTS13 haplotypes are associated with differential expression and activity of the VWF-cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family.
Kim B, Hing ZA, Wu A, Schiller T, Struble EB, Liuwantara D, Kempert PH, Broxham EJ, Edwards NC, Marder VJ, Simhadri VL, Sauna ZE, Howard TE, Kimchi-Sarfaty C

Haemophilia 2014 Mar;20(2):e157-63
Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions.
Simhadri VL, Hamasaki-Katagiri N, Tseng SC, Bentley AA, Zichel R, Hershko AY, Komar AA, Kimchi-Sarfaty C

Cancer Res 2014 Jan 15;74(2):598-608
MDR1 synonymous polymorphisms alter transporter specificity and protein stability in a stable epithelial monolayer.
Fung KL, Pan J, Ohnuma S, Lund PE, Pixley JN, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM

Nat Med 2013 Oct;19(10):1318-24
Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A.
Pandey GS, Yanover C, Miller-Jenkins LM, Garfield S, Cole SA, Curran JE, Moses EK, Rydz N, Simhadri V, Kimchi-Sarfaty C, Lillicrap D, Viel KR, Przytycka TM, Pierce GF, Howard TE, Sauna ZE, the PATH (Personalized Alternative Therapies for Hemophilia) Study Investigators

Trends Pharmacol Sci 2013 Oct;34(10):534-48
Building better drugs: developing and regulating engineered therapeutic proteins.
Kimchi-Sarfaty C, Schiller T, Hamasaki-Katagiri N, Khan MA, Yanover C, Sauna ZE

Proc Natl Acad Sci U S A 2013 Aug 13;110(33):13481-6
Whole-genome sequencing identifies a recurrent functional synonymous mutation in melanoma.
Gartner JJ, Parker SC, Prickett TD, Dutton-Regester K, Stitzel ML, Lin JC, Davis S, Simhadri VL, Jha S, Katagiri N, Gotea V, Teer JK, Wei X, Morken MA, Bhanot UK; NISC Comparative Sequencing Program, Chen G, Elnitski LL, Davies MA, Gershenwald JE, Carter H, Karchin R, Robinson W, Robinson S, Rosenberg SA, Collins FS, Parmigiani G, Komar AA, Kimchi-Sarfaty C, Hayward NK, Margulies EH, Samuels Y

J Mol Biol 2013 Nov 1;425(21):4023-33
A gene-specific method for predicting hemophilia-causing point mutations.
Hamasaki-Katagiri N, Salari R, Wu A, Qi Y, Schiller T, Filiberto AC, Schisterman EF, Komar AA, Przytycka TM, Kimchi-Sarfaty C

Br J Haematol 2013 Mar;160(6):825-37
Multiple in silico tools predict phenotypic manifestations in congenital thrombotic thrombocytopenic purpura.
Hing ZA, Schiller T, Wu A, Hamasaki-Katagiri N, Struble EB, Russek-Cohen E, Kimchi-Sarfaty C

Nucleic Acids Res 2013 Jan 1;41(1):44-53
Sensitive measurement of single-nucleotide polymorphism-induced changes of RNA conformation: application to disease studies.
Salari R, Kimchi-Sarfaty C, Gottesman MM, Przytycka TM

J Biol Chem 2012 Dec 28;287(53):44361-71
Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).
Hershko K, Simhadri V, Blaisdell A, Hunt RC, Newell J, Tseng SC, Hershko AY, Choi JW, Sauna ZE, Wu A, Bram RJ, Komar AA, Kimchi-Sarfaty C

Haemophilia 2012 Nov;18(6):933-40
Analysis of F9 point mutations and their correlation to severity of haemophilia B disease.
Hamasaki-Katagiri N, Salari R, Simhadri VL, Tseng SC, Needlman E, Edwards NC, Sauna ZE, Grigoryan V, Komar AA, Przytycka TM, Kimchi-Sarfaty C

PLoS One 2012;7(6):e38864
Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches.
Edwards NC, Hing ZA, Perry A, Blaisdell A, Kopelman DB, Fathke R, Plum W, Newell J, Allen CE, S G, Shapiro A, Okunji C, Kosti I, Shomron N, Grigoryan V, Przytycka TM, Sauna ZE, Salari R, Mandel-Gutfreund Y, Komar AA, Kimchi-Sarfaty C

Biologicals 2012 May;40(3):191-5
Plasma derivatives: new products and new approaches.
Sauna ZE, Pandey GS, Jain N, Mahmood I, Kimchi-Sarfaty C, Golding B

Nat Rev Genet 2011 Oct;12(10):683-91
Understanding the contribution of synonymous mutations to human disease.
Sauna ZE, Kimchi-Sarfaty C

Pharmacogenomics 2011 Aug;12(8):1147-60
Tseng SC, Kimchi-Sarfaty C

Mol Biosyst 2011 Jun;7(6):2012-8
Detection of a secreted metalloprotease within the nuclei of liver cells.
Hunt RC, Geetha S, Allen CE, Hershko K, Fathke R, Kong PL, Plum E, Struble EB, Soejima K, Friedman S, Garfield S, Balaji S, Kimchi-Sarfaty C

PLoS One 2011 Mar 22;6(3):e17981
Inhibition of Multidrug Resistance by SV40 Pseudovirion Delivery of an Antigene Peptide Nucleic Acid (PNA) in Cultured Cells.
Macadangdang B, Zhang N, Lund PE, Marple AH, Okabe M, Gottesman MM, Appella DH, Kimchi-Sarfaty C

Thromb Haemost 2010 Sep;104(3):531-5
A splice variant of ADAMTS13 is expressed in human hepatic stellate cells and cancerous tissues.
Shomron N, Hamasaki-Katagiri N, Hunt R, Hershko K, Pommier E, Geetha S, Blaisdell A, Marple A, Roma I, Newell J, Allen C, Friedman S, Kimchi-Sarfaty C

Pharm Res 2010 Mar;27(3):400-20
Pseudovirions as vehicles for the delivery of siRNA.
Lund PE, Hunt RC, Gottesman MM, Kimchi-Sarfaty C

Methods Mol Biol 2009;578:23-39
Silent (Synonymous) SNPs: Should We Care About Them?
Hunt R, Sauna ZE, Ambudkar SV, Gottesman MM, Kimchi-Sarfaty C

Cytometry A 2009 Aug;75(8):675-81
Detection of intracellular ADAMTS13, a secreted zinc-metalloprotease, via flow cytometry.
Geetha S, Allen CE, Hunt RC, Plum E, Garfield S, Friedman SL, Soejima K, Sauna ZE, Kimchi-Sarfaty C

PLoS One 2009 Aug 5;4(8):e6506
Characterization of conformation-sensitive antibodies to ADAMTS13, the von Willebrand cleavage protease.
Sauna ZE, Okunji C, Hunt RC, Gupta T, Allen CE, Plum E, Blaisdell A, Grigoryan V, Geetha S, Fathke R, Soejima K, Kimchi-Sarfaty C

J Mol Biol 2008 Nov 7;383(2):281-91
Synonymous mutations and ribosome stalling can lead to altered folding pathways and distinct minima.
Tsai CJ, Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM, Nussinov R

Mol Pharmacol 2008 Apr;73(4):1254-63
Modulation of Na+-Ca2+ exchanger expression by immunosuppressive drugs is isoform-specific.
Elbaz B, Alperovitch A, Gottesman MM, Kimchi-Sarfaty C, Rahamimoff H

Cancer Res 2007 Oct 15;67(20):9609-12
Silent polymorphisms speak: how they affect pharmacogenomics and the treatment of cancer.
Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM

Pharmacogenomics 2007 Jun;8(6):527-32
The sounds of silence: synonymous mutations affect function.
Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM

Ann N Y Acad Sci 2007 Mar;1099:204-14
Cyclosporin A-dependent downregulation of the Na+/Ca2+ exchanger expression.
Rahamimoff H, Elbaz B, Alperovich A, Kimchi-Sarfaty C, Gottesman MM, Lichtenstein Y, Eskin-Shwartz M, Kasir J

Science 2007 Jan 26;315(5811):525-8
A "Silent" Polymorphism in the MDR1 Gene Changes Substrate Specificity.
Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, Gottesman MM

Pharmacogenomics 2007 Jan;8(1):29-39
Ethnicity-related polymorphisms and haplotypes in the human ABCB1 gene.
Kimchi-Sarfaty C, Marple AH, Shinar S, Kimchi AM, Scavo D, Roma MI, Kim IW, Jones A, Arora M, Gribar J, Gurwitz D, Gottesman MM

Cancer Gene Ther 2006 Jul;13(7):648-57
SV40 Pseudovirion gene delivery of a toxin to treat human adenocarcinomas in mice.
Kimchi-Sarfaty C, Vieira WD, Dodds D, Sherman A, Kreitman RJ, Shinar S, Gottesman MM

Hum Gene Ther 2005 Sep;16(9):1110-5
Efficient Delivery of RNA Interference Effectors via In Vitro-Packaged SV40 Pseudovirions.
Kimchi-Sarfaty C, Brittain S, Garfield S, Caplen NJ, Tang Q, Gottesman MM



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