Gene therapies for neurodegenerative disease

An active, viral vector technology for use in gene therapy for neurodegenerative diseases, addressing issues such as limited treatment options

Pending Publication Date: 2020-10-23
普利维尔治疗公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Currently, treatment optio

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  • Gene therapies for neurodegenerative disease
  • Gene therapies for neurodegenerative disease
  • Gene therapies for neurodegenerative disease

Examples

Experimental program
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Embodiment 1

[0110] Example 1: rAAV vector

[0111] AAV vectors are produced using cells such as HEK293 cells for triple plasmid transfection. ITR sequences flank expression constructs containing promoter / enhancer elements, 3' polyA signals, and post-translational signals such as WPRE elements for each transgene of interest. Multiple gene products, such as C9orf72 protein or GBA1 protein, and one or more inhibitory nucleic acids (e.g., inhibitory nucleic acids targeting C9orf72 and / or TMEM106B) can be expressed simultaneously, e.g., by using a single expression cassette or separate expression cassettes Express. The presence of highly spliced ​​short intronic sequences upstream of the expressed gene can increase expression levels. Inhibitory RNAs (eg, miRNAs, shRNAs, etc.) and other regulatory RNAs can potentially be included in these sequences. Examples of expression constructs described in this disclosure are shown in Figure 1-17 and 20-21 and in Table 1 below.

[0112] Table 1

[...

Embodiment 2

[0115] Example 2: Cell-Based Assay for Viral Transduction in ALS / FTD Cells

[0116] Cells characterized by the repeat expansion of C9orf72 are obtained as, for example, fibroblasts, monocytes or hES cells from ALS / FTD patients or patient-derived induced pluripotent stem cells (iPSCs). These cells accumulate RNA foci and proteins translated from RAN.

[0117] Using these cell models, cellular disease is quantified based on the accumulation of protein aggregates, such as aggregates of the RAN protein, using anti-RAN protein antibodies, followed by fluorescence microscopy imaging. Abnormal accumulation of RAN protein was quantified using Western blot and / or ELISA.

[0118] Therapeutic endpoints (eg, reduction of ALS / FTD-associated disease) are measured in the context of transduced expression of AAV vectors to confirm and quantify activity and function. Reduction in endogenous (eg, pathogenic repeat expansion-containing) C9orf72 mRNA levels can be quantified, eg, using quantitat...

Embodiment 3

[0119] Example 3: In vitro studies

[0120] This example describes in vitro testing of the C9orf72 rAAV vector described in this disclosure. The effects of C9orf72 knockdown and overexpression were studied in mammalian cells. Examples of constructs tested are listed in Table 2.

[0121] ID Promoter knock down Promoter Overexpression I00017 H1 C9_sh CMV opt-C9 I00018 CMV C9_sh, TMEM_mi CMV opt-C9 I00030 CMV_intronic C9_sh(cons) CMV opt-C9 I00031 CMV_intronic C9repeat_sh CMV opt-C9 I00032 CMV_intronic C9_sh (validated) CMV opt-C9 I00037 CMV_intronic C9r_sh, ATXN_sh CMV opt-C9

[0122] Gene knockdown and overexpression were determined by quantitative PCR (qPCR) and ELISA. Figure 19A Representative data shown demonstrates statistically significant silencing of C9orf72 by rAAV vectors. Figure 19B Representative data shown demonstrates a statistically significant increase in wild-type C...

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Abstract

The disclosure relates, at some aspects, to compositions and methods for treatment of neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), Alzheimer's disease, Gaucher disease, Parkinson's disease, Lewy body dementia, or a lysosomal storage disease). In some embodiments, the disclosure provides expression constructs comprising a transgene encoding one or more inhibitory nucleic acids (e.g., targeting C9orf72, TMEM106B, ATNX2, RPS25, etc.), wild-type C9orf72 protein or a portion thereof, or any combination of the foregoing. In some embodiments, the disclosure provides methods of ALS/FTD by administering such expression constructs to a subject in need thereof.

Description

[0001] related application [0002] This application claims U.S. Provisional Application Serial No. 62 / 742,723, filed October 8, 2018, entitled "GENETHERAPIES FOR NEURODEGENERATIVE DISEASE," and filed October 23, 2017, entitled " 62 / 575,795 of the interest under 35 U.S.C. 119(e) of GENE THERAPIES FORNEURODE GENERATIVE DISEASE, the entire contents of each of which provisional applications are incorporated herein by reference. Background technique [0003] In humans, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases associated with expansion of the hexanucleotide repeat region in the C9orf72 gene. Typically, diseases associated with expansion of the C9orf72 repeat region result from decreased expression of C9orf72 protein and gain of function due to accumulation of toxic RNA foci. Currently, treatment options for ALS / FTD are limited. Contents of the invention [0004] Aspects of the present disclosure relate to neurodegenera...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/86
CPCC12N15/113C12N15/86C12N7/00A61K31/7105A61P25/28A61P25/16A61P25/00C12N2750/14143C12N2750/14121C12N2710/14043C12N2750/14152C12N2830/48C12N2830/50C12N2310/14C12N2320/32C12N15/1138C12N2330/51C12N2310/531A61K9/0085
Inventor 阿萨·阿贝利奥维奇劳拉·赫克曼赫维·莱茵
Owner 普利维尔治疗公司
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