A leading RNAi therapy company, Alnylam, recently disclosed its performance results for 2023, which revealed that its two siRNA drugs targeting transthyretin (TTR) for the treatment of amyloidosis, patisiran and vutrisiran, collectively generated sales exceeding $913 million [1] . Globally, there are between 250,000 to 550,000 patients with transthyretin amyloidosis (ATTR), with a larger number suffering from ATTR cardiomyopathy (ATTR-CM) [2] . However, patisiran and vutrisiran are currently approved only for the treatment of ATTR polyneuropathy (ATTR-PN). Consequently, Alnylam is conducting clinical trials to evaluate the efficacy of patisiran and vutrisiran in treating ATTR-CM. Concurrently, numerous pharmaceutical companies are also developing TTR-targeted therapies, including small nucleic acids, small molecules, antibodies, or gene editing therapies—demonstrating the intensifying competition in the development of TTR-targeting drugs for the treatment of ATTR.

Figure 1: Pathogenesis and Clinical Manifestations of Transthyretin Amyloidosis (ATTR) [3] .

The TTR Protein and Transthyretin Amyloidosis (ATTR)

The most common type of hereditary amyloidosis is ATTR, a lethal disease caused by the misfolding of transthyretin protein (TTR) from pathogenic changes (or mutations) in the genetic code. In its normal state, TTR protein exists in the form of tetramers in the peripheral blood, responsible for transporting thyroxine and retinol-binding protein. In ATTR patients, TTR protein misfolds and aggregates into amyloid-like substances, which deposit in tissues and organs, leading to organ dysfunction, failure, and even death [4] . Based on the presence of mutations in the TTR gene, ATTR can be classified into hereditary/mutant type (ATTRv) and non-hereditary/wild-type (ATTRwt) [5] .

Figure 2: Differential Incidence and Clinical Presentations of ATTRwt and ATTRv [6] . 

ATTRv is caused by pathogenic genetic mutations in the TTR gene and can occur at any age between 20 and 80 years old. These mutations lead to the dissociation of TTR protein from its tetrameric state into monomers, which then misfold and aggregate into amyloid-like substances, ultimately accumulating in organs such as the heart, nerves, and kidneys. In contrast, ATTRwt does not involve mutations in the TTR gene but arises from the failure of age-related homeostasis mechanisms. With influences from factors caused by aging, wild-type TTR protein may undergo misfolding similar to that seen in mutant-type TTR protein. ATTRwt is a slowly progressive disease that typically manifests after the age of 65, so it has also been known as senile systemic amyloidosis.

Targeting the TTR Gene and Protein Therapy for ATTR

Therapeutic drug development for ATTR primarily focuses on inhibiting the synthesis of TTR protein, stabilizing the tetrameric structure of TTR protein, or clearing misfolded TTR protein [7] . Among these approaches, small nucleic acids and gene editing are prominent methods. Several antisense oligonucleotide (ASO) and small interfering RNA (siRNA) drugs have been demonstrated to effectively suppress the synthesis of mutant and/or wild-type TTR protein, improving the disease phenotype in patients, with many of these drugs successfully approved and marketed. Additionally, therapies utilizing gene editing techniques such as CRISPR have shown promising early clinical results in reducing TTR gene expression [8] .


Figure 3: Various Innovative Therapies Targeting the TTR Gene and Protein for the Treatment of ATTR [8] . 

Both small nucleic acid and gene editing therapies target human genes for modification. For example, Patisiran aims to target and silence the 3'UTR of TTR mRNA, thereby blocking the production of TTR protein. Therefore, the humanization of mouse genes is essential for developing animal models that will provide the most effective platforms for preclinical studies. Cyagen has developed two humanized mouse models of the mouse Ttr gene, replacing it with the full human gene sequence and the equivalent pathogenic human gene mutation (p.V50M) model:

  • The B6-hTTR mouse (Product ID: C001512), expressing the human wild-type TTR gene.
  • The H11-Alb-hTTR*V50M mouse (Product ID: C001525), which overexpresses the human TTR gene carrying the p.V50M pathogenic mutation.

Both models can meet the requirements for most gene editing, ASO, and siRNA therapies in the treatment research of ATTRwt and ATTRv. Below are detailed descriptions of these next-generation humanized mouse models.

The B6-hTTR Mice Successfully Express Human TTR Gene and Protein

RT-qPCR and ELISA detection show that B6-hTTR mice successfully express humanized TTR gene and protein, and there is no expression of endogenous mouse Ttr gene.

Figure 4: Detection of Humanized TTR Gene and Protein Expression in B6-hTTR Mice

B6-hTTR Mice Are Utilized For The Efficacy Evaluation Of Small Nucleic Acid Drugs

Vutrisiran, an siRNA drug that can bind to and degrade both wild-type and mutant human TTR mRNA, received approval in 2022 for the treatment of adult hereditary ATTR-PN [9] . Validation data indicates that after a single subcutaneous injection of Vutrisiran, the expression levels of human TTR protein in the plasma of B6-hTTR mice are significantly lower compared to the control group.

Figure 5: In Vivo Efficacy Testing of Small Nucleic Acid Drug Vutrisiran in B6-hTTR Mice

The H11-Alb-hTTR*V50M Mice Express Human Mutant TTR Gene and Protein

The H11-Alb-hTTR*V50M mice were constructed by inserting the human TTR gene carrying the p.V50M pathogenic mutation into the mouse H11 safe harbor locus. This model successfully expresses humanized TTR gene and protein, and its expression levels in vivo far exceed those of B6-hTTR mice.

Figure 6: Detection of Humanized TTR Gene and Protein Expression in H11-Alb-hTTR*V50M Mice

In Summary

B6-hTTR mice (Product ID: C001512) and H11-Alb-hTTR*V50M mice (Product ID: C001525) both successfully express humanized TTR gene and protein. The TTR mRNA in these mouse models can be effectively targeted by small nucleic acid drugs targeting human TTR, thereby inhibiting its translation expression and reducing the levels of humanized TTR protein in mice. Therefore, these two mouse models serve as effective tools for studying the mechanisms of ATTR disease and evaluating the efficacy of targeted drugs, especially in the development of precise targeting therapies such as CRISPR gene editing, antisense oligonucleotides (ASOs), or small interfering RNA (siRNA) drugs that target human TTR gene and mRNA.

Furthermore, Cyagen utilizes its proprietary TurboKnockout fusion BAC recombination technology to offer customized services based on the B6-hTTR mouse model. This technology enables the construction of other popular humanized point mutation disease models, tailored to different point mutations, to meet the needs of researchers for drug screening and pharmacological experiments aimed at targeting various TTR pathogenic mutations with precision therapies.



References:

[1]Alnylam Pharmaceuticals, Inc. (2024, February 15). Alnylam Pharmaceuticals Reports Fourth Quarter and Full Year 2023 Financial Results and Highlights Recent Period Activity [Press release]. Retrieved from https://investors.alnylam.com/press-release?id=27941

[2]Gillmore JD, Gane E, Taubel J, Kao J, Fontana M, Maitland ML, Seitzer J, O'Connell D, Walsh KR, Wood K, Phillips J, Xu Y, Amaral A, Boyd AP, Cehelsky JE, McKee MD, Schiermeier A, Harari O, Murphy A, Kyratsous CA, Zambrowicz B, Soltys R, Gutstein DE, Leonard J, Sepp-Lorenzino L, Lebwohl D. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N Engl J Med. 2021 Aug 5;385(6):493-502.

[3]Ibrahim RB, Liu YT, Yeh SY, Tsai JW. Contributions of Animal Models to the Mechanisms and Therapies of Transthyretin Amyloidosis. Front Physiol. 2019 Apr 2;10:338. 

[4]Nativi-Nicolau JN, Karam C, Khella S, Maurer MS. Screening for ATTR amyloidosis in the clinic: overlapping disorders, misdiagnosis, and multiorgan awareness. Heart Fail Rev. 2022 May;27(3):785-793.

[5]Koike H, Katsuno M. Transthyretin Amyloidosis: Update on the Clinical Spectrum, Pathogenesis, and Disease-Modifying Therapies. Neurol Ther. 2020 Dec;9(2):317-333.

[6]Merino-Merino AM, Labrador-Gomez J, Sanchez-Corral E, Delgado-Lopez PD, Perez-Rivera JA. Utility of Genetic Testing in Patients with Transthyretin Amyloid Cardiomyopathy: A Brief Review. Biomedicines. 2023 Dec 21;12(1):25.

[7]Aimo A, Castiglione V, Rapezzi C, Franzini M, Panichella G, Vergaro G, Gillmore J, Fontana M, Passino C, Emdin M. RNA-targeting and gene editing therapies for transthyretin amyloidosis. Nat Rev Cardiol. 2022 Oct;19(10):655-667. 

[8]Tomasoni D, Bonfioli GB, Aimo A, Adamo M, Canepa M, Inciardi RM, Lombardi CM, Nardi M, Pagnesi M, Riccardi M, Vergaro G, Vizzardi E, Emdin M, Metra M. Treating amyloid transthyretin cardiomyopathy: lessons learned from clinical trials. Front Cardiovasc Med. 2023 May 23;10:1154594. 

[9]Keam SJ. Vutrisiran: First Approval. Drugs. 2022 Sep;82(13):1419-1425. 

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