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Frataxin expression constructs having engineered promoters and methods of use thereof

A promoter and engineering technology, applied in the field of mutaxin-based compositions, can solve problems such as ineffective treatment of FA

Pending Publication Date: 2021-09-10
VOYAGER THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no effective treatment for FA, and patients are usually only monitored for symptom management

Method used

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  • Frataxin expression constructs having engineered promoters and methods of use thereof
  • Frataxin expression constructs having engineered promoters and methods of use thereof
  • Frataxin expression constructs having engineered promoters and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0857] Example 1. Design of promoter variants

[0858] A. Promoter

[0859] Previous studies have shown that the CMV promoter drives the highest level of cotaxin expression, whereas CBA shows less efficacy in driving expression. The PGK and FXN promoters have been shown to be even weaker promoters for kinesin expression. Variants of the CMV, CBA and FXN promoters were generated to determine which promoters would result in the highest expression of payloads such as frataxin or luciferase. The promoter is inserted into a luciferase expressing vector constructed using standard molecular cloning techniques.

[0860] The designed promoters are described in Table 3 above. In Table 3, CMV stands for "cytomegalovirus", CBA stands for "chicken β-actin" possibly with CMV IE enhancer region and promoter region, CAG stands for CMV enhancer, CBA promoter and rabbit β-bead Protein splice acceptor site, FXN stands for "fetaxin" and mCBA stands for a variant of the CBA promoter generate...

Embodiment 2

[0876] Example 2. Design of payload constructs encoding frataxin

[0877] The payload construct is designed to contain minimal nucleic acid sequence encoding frataxin. Build payload constructs using standard molecular cloning techniques. The FXN tag transgene was cloned into an AAV expression vector and the resulting clone was further sequenced to confirm the correctness of all elements such as ITR, promoter and tag.

[0878] To construct the cynomolgus monkey futaxin (cFXN) payload construct, a hemagglutinin (HA)-tagged cFXN transgene was cloned into a gene containing 5′ and 3′ ITR sequences (141 nucleotides) derived from the AAV2 genome, CBA, CMV or FXN promoter, hβ globin intron region, hGH poly(A) signal and three miR-122 binding sites (miR-122BS) for liver-detargeting in plasmids. Deletion variants of the CBA, CMV or FXN promoters were evaluated. A 450 bp human albumin sequence (Alb450) was tested as a filler sequence in three constructs (see Table 4; ITR to ITR sequ...

Embodiment 3

[0882] Example 3. Validation of cFXN constructs and components thereof

[0883] A. Identification of promoter variants and hβ globin introns

[0884] To verify the promoter and hβ globin intron regions in engineered cFXN constructs driven by CMV, CBA, and FXN promoter variants, high-fidelity restriction enzymes MluI-HF and AgeI-HF Digest the construct. Double digestion yields a cleavage product consisting of the promoter and hβ globin intron regions. Digested samples were analyzed by agarose gel electrophoresis. Variants tested included cFXN1-cFXN18 (SEQ ID NO: 1778-1795). The gel shows bands with a pattern corresponding to the size of the promoter variant.

[0885] B. Identification of miR-122 and hGH poly(A)

[0886] To validate miR-122 and hGH poly(A) regions in engineered cFXN constructs driven by CMV, CBA and FXN promoter variants, the constructs were digested with restriction enzymes XhoI and ClaI. Double digestion yielded a cleavage product consisting of the miR...

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Abstract

The disclosure relates to compositions and methods for altering, e.g., enhancing, the expression of frataxin (FXN), whether in vitro and / or in vivo including, but not limited to, the exploitation of engineered promoters. Such compositions include delivery via administration of an adeno-associated viral (AAV) particle. The compositions and methods of the present disclosure are useful in the treatment of subjects diagnosed with, or suspected of having Friedreich's ataxia or another neuromuscular or neurological condition resulting from a deficiency in the quantity and / or function of frataxin or associated with decreased expression or protein levels of frataxin.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Patent Application No. 62 / 738,519, filed September 28, 2018, entitled "Mataxin Compositions and Methods of Using Same," and filed September 17, 2019, and entitled "Having The benefit of U.S. Provisional Patent Application No. 62 / 901,769 of Mataxin Expression Constructs with Engineered Promoters and Methods of Using the Same, the contents of which are incorporated herein by reference in their entirety. [0003] Reference Sequence Listing [0004] This application is filed with a sequence listing in electronic format. The sequence listing file name is 20571019PCTSEQLST.txt, the file was created on September 27, 2019, and the size is 6,732,273 bytes. The information in electronic format of the Sequence Listing is hereby incorporated by reference in its entirety. [0005] field of invention [0006] The present invention relates to frataxin-based compositions and methods that involv...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K31/7088A61K48/00
CPCA61K48/005C07K14/47A61K9/0019A61K9/08A61K47/10A61K47/02C12N2750/14143A61K48/0075A61K48/0058C12N2830/008C12N15/86A61P25/00A61P21/00
Inventor H.帕茨克J.侯H.王Y.舒M.古莱特D.W-Y.萨
Owner VOYAGER THERAPEUTICS
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