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: OBRR / DH / LH

General Overview

Recombinant proteins (proteins made using genetic engineering) that are used to treat blood clotting disorders have several advantages over human plasma-derived products. Unlike plasma-derived products, which must be harvested from the pool of donor plasma that is available, recombinant proteins can be manufactured cost-effectively, with decreased risk of viral contamination and with increased product uniformity and flexibility in product design. However, the benefit that this flexibility provides presents a new challenge for FDA regulators. This challenge is based on the genetic code that controls the production of each type of protein.

Manufacturers of recombinant proteins must choose the gene sequence of the therapeutic recombinant protein. This sequence, which is the order in which specific nucleic acid building blocks of the gene are arranged, is one of the factors that determine the shape of the protein and how it works in the body. However, the reality is that there is variability in this sequence among different people. In addition to the common form of a given protein (termed wild-type), genetic variations exist from one individual to the next. These variations can significantly affect the protein's function, how quickly the body breaks it down, where it goes in the body, its immunogenicity (how readily it triggers an immune response) and other important therapeutic properties. Currently, FDA does not stringently regulate the manufacturer's choice of a gene sequence for manufacturing a therapeutic protein. Therefore, our research program was formed in order to help FDA develop guidelines for choosing such gene sequences by improving our understanding of the impact of the variation in gene sequences on the safety and effectiveness of therapeutic recombinant proteins when administered to humans.

Our program studies the pharmacogenomics of clotting factors (genetic variations of protein therapeutics that affect their characteristics). We chose two proteins as prototypes to study using a combination of in silico (computer analysis) and in vitro ("test tube") experiments in the laboratory: ADAMTS13 and factor IX (FIX).

ADAMTS13 is an anti-clotting factor, a protein that breaks down blood clots after the injury is healed. The gene for this protein was only recently discovered. Factor IX (FIX), which helps to trigger blood clotting, has been extensively studied over the past few decades. Absence of FIX causes hemophilia, and there are FDA-approved plasma-derived and recombinant products based on this protein.

We are developing sensitive new assays that distinguish among slightly different forms of each protein. These tools can also distinguish among variations in these proteins caused by differences in their gene sequences. Such differences may be Single Nucleotide Polymorphisms (SNPs), a DNA variation that occurs in at least 1% of the population, or splicing forms (DNA pieces that are cut and reconnected into codes for different forms of the same protein). Similarly, these variations may be on the protein level, such as additional of sugar molecules.

These assays will help researchers and product manufacturers to determine the expression, function, and conformation (shape) of ADAMTS13, FIX, and, eventually, other clotting factors.

We are also establishing computer-based systems that compare genetic variations in order to predict and measure the effects of gene sequence on the properties of proteins.

Finally, to add to the knowledge base of ADAMTS13, we are investigating its life cycle and its interaction with other clotting factors and immunosuppressive drugs (drugs that suppress immune function, like those used to prevent transplant rejection). The goal of our studies is to add to our current understanding of blood clotting proteins and to support the development of regulatory oversight of these products to ensure that they are safe and effective.

Scientific Overview

The number of therapeutic applications for recombinant proteins, particularly for clotting factors continues to grow. In order to facilitate cloning and expression, industry has being developing recombinant proteins with synonymous or non-synonymous mutation-harboring sequences. However, there is currently no guidance or protocol for the industry regarding the genetic sequence used to manufacture recombinant proteins. However, FDA requires verification that the sequence in the working cell bank exactly matches that used for transfection in original cell lines. Thus, FDA is at a critical juncture as it becomes increasingly clear that guidelines must be developed for determining and validating the efficiency and safety of recombinant protein and the sequences that encode them.

It is a common assumption that "silent" mutations in genes do not affect the expression, functionality, half-life or immunogenicity of the proteins they encode. In addition, it is frequently believed that a therapeutic protein harboring common, single nucleotide polymorphisms can be used to treat all types of patients. We are investigating this assumption in order to ensure that FDA has the information needed to develop and institute appropriate regulations. We anticipate that in silico criteria for narrowing sequencing options will reduce the cost and time required to develop recombinant proteins and increase the safety and efficacy of these proteins.

Our investigation of the splicing forms, synonymous mutations, and various polymorphisms of recombinant proteins will help to determine how drug manufacturers should engineer the gene sequences upon which their products are based. Moreover, the testing methods we create to monitor gene pathways and protein expression and function will facilitate the development of standard testing protocols for validating the safety and efficacy of various therapeutic proteins. At present our preliminary results in investigating polymorphisms and mutations in Factor IX (FIX) have indicated a new cause of hemophilia B. In addition, we demonstrated that several synonymous polymorphisms in ADAMTS13 increase or decrease the specific activity and change the conformation of this molecule. Findings such will benefit the public and scientific community in the diagnosis and treatment of patients.

As part of this investigation, we have established the following methods to monitor the influence of alternative splicing forms, single mutations, and polymorphisms on FIX and ADAMTS13: (1) mRNA (qRT-PCR); (2) intracellular expression (flow cytometry and Western blotting); (3) protein conformation (comparing staining patterns of various antibodies on reducing and non-reducing gels (using and developing conformation-sensitive antibodies, and analysis of trypsin digestion patterns); (4) protein secretion (reducing and non-reducing conditions in the presence or absence of proteasome and lysosome inhibitors); and (5) extracellular function (multiple activity assays)

We have chosen to demonstrate this principle in the context of ADAMTS13 (the von Willebrand factor-cleaving protease) and FIX.

Our major contributions to the field of clotting factor pharmacogenomics can be summarized as follows: (1) establishment of a panel of conformation-sensitive antibodies capable of differentiating ADAMTS13 based on catalytic ability (currently working on such panel for FVIII); (2) demonstration that mutations, splicing forms, or polymorphisms, both synonymous and non-synonymous, can modify expression and or function of recombinant proteins; and (3) characterization of ADAMTS13 (intra- and extracellular forms), its possible interaction with FVIII, and its possible translocation to the nucleus—all of which have potential clinical and regulatory relevance.


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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