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  5. Toward More Effective Treatment of Blood Clotting Disorders: Pharmacogenomic Studies of ADAMTS13 and Related Proteins
  1. Biologics Research Projects

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.


Int J Mol Sci 2019 Nov 15;20(22)
A single synonymous variant (c.354G>A [p.P118P]) in ADAMTS13 confers enhanced specific activity.
Hunt R, Hettiarachchi G, Katneni U, Hernandez N, Holcomb D, Kames J, Alnifaidy R, Lin B, Hamasaki-Katagiri N, Wesley A, Kafri T, Morris C, Bouche L, Panico M, Schiller T, Ibla J, Bar H, Ismail A, Morris H, Komar A, Kimchi-Sarfaty C

Sci Rep 2019 Oct 29;9(1):15449
Effects of codon optimization on coagulation factor IX translation and structure: implications for protein and gene therapies.
Alexaki A, Hettiarachchi GK, Athey JC, Katneni UK, Simhadri V, Hamasaki-Katagiri N, Nanavaty P, Lin B, Takeda K, Freedberg D, Monroe D, McGill JR, Peters R, Kames JM, Holcomb DD, Hunt RC, Sauna ZE, Gelinas A, Janjic N, DiCuccio M, Bar H, Komar AA, Kimchi-Sarfaty C

Infect Genet Evol 2019 Sep;73:266-8
The Kazusa codon usage database, CoCoPUTs, and the value of up-to-date codon usage statistics.
Holcomb DD, Alexaki A, Katneni U, Kimchi-Sarfaty C

Mol Genet Genomic Med 2019 Aug;7(8):e840
Splicing dysregulation contributes to the pathogenicity of several F9 exonic point variants.
Katneni UK, Liss A, Holcomb D, Katagiri NH, Hunt R, Bar H, Ismail A, Komar AA, Kimchi-Sarfaty C

Am J Physiol Gastrointest Liver Physiol 2019 Jun 1;316(6):G720-34
Translational and transcriptional responses in human primary hepatocytes under hypoxia.
Hettiarachchi GK, Katneni UK, Hunt RC, Kames JM, Athey JC, Bar H, Sauna ZE, McGill JR, Ibla JC, Kimchi-Sarfaty C

J Mol Biol 2019 Jun 14;431(13):2434-41
Codon and codon-pair usage tables (CoCoPUTs): facilitating genetic variation analyses and recombinant gene design.
Alexaki A, Kames J, Holcomb DD, Athey J, Santana-Quintero LV, Lam PVN, Hamasaki-Katagiri N, Osipova E, Simonyan V, Bar H, Komar AA, Kimchi-Sarfaty C

J Thromb Haemost 2019 Mar;17(3):429-40
Von Willebrand Factor/ADAMTS-13 interactions at birth: implications for thrombosis in the neonatal period.
Katneni UK, Ibla JC, Hunt R, Schiller T, Kimchi-Sarfaty C

J Thromb Haemost 2017 Dec;15(12):2306-16
Elevated preoperative von Willebrand Factor is associated with perioperative thrombosis in infants and neonates with congenital heart disease.
Hunt R, Hoffman CM, Emani S, Trenor CC 3rd, Emani SM, Faraoni D, Kimchi-Sarfaty C, Ibla JC

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

Blood Adv 2017 Jun 19;1(15):1037-46
Genetic variants in ADAMTS13 as well as smoking are major determinants of plasma ADAMTS13 levels.
Ma Q, Jacobi PM, Emmer BT, Kretz CA, Ozel AB, McGee B, Kimchi-Sarfaty C, Ginsburg D, Li JZ, Desch KC

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

Per 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

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