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Carrier

A carrier and precursor technology, applied in the direction of carriers, nucleic acid carriers, and the use of carriers to introduce foreign genetic material, can solve problems such as expensive

Pending Publication Date: 2022-05-06
伦敦王室学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, AAV vector production is an extremely expensive process, currently estimated at ~$1 million per patient

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1: Improve the efficiency of PGRN translation by codon optimization algorithm

[0155] The rate of protein translation from RNA transcripts can be increased by changing codons to use codons that are optimal for a particular species, and gene expression in a particular cell type can be increased. By optimizing protein production per transcript, fewer viral vectors are used, reducing toxicity risk and cost. Optimization of wild-type PGRN transcripts was modeled using three open source programs (IDT, Geneart and Genscript: see Materials and methods for details). The codon adaptation index (CAI; https: / / www.genscript.com / tools / rare-codon-analysis) was validated using the rare codon analysis tool. A CAI of 1.0 was considered ideal, while a CAI >0.8 was rated as good expression in the target expressing organism. The lower the number, the higher the likelihood of poor gene expression.

[0156] 1.1 Codon optimization design

[0157] Using a commercially available co...

Embodiment 2

[0192] Example 2: Development of intron enhancing elements to increase PGRN expression.

[0193] To further increase the expression and secretion of PGRN, the addition of an intron in the 5′ sequence of PGRN was explored to see if gene expression could be enhanced. Introns can increase transcription levels by affecting transcription rate, nuclear export, transcript stability, or mRNA translation efficiency, a phenomenon known as intron-mediated enhancement (IME) ( Shaul O. Int J Biochem Cell Biol. 2017 Oct;91(Pt B):145-155). A search was performed for intron sequences smaller than 300 bp that could be used to test for intron-mediated enhancement. Introns within the human growth hormone (hGH) gene were selected as suitable candidates (see Figure 5 a).

[0194] The wild-type GRN sequence was genetically synthesized and cloned into pAAV plasmid (Addgene; 99280) using BamH1 and Xho1 restriction sites. These constructs were used as a master vector (pAAV-CMV-PGRNwt) to sub-clon...

Embodiment 3

[0216] Example 3: Combining intronic and exonic enhancer elements.

[0217] To test the intron-mediated enhancement of codon-optimized PGRN-GS by hGHi3, the hGHi3 element was subcloned into the 5' position of PGRN-GS.

[0218] Surprisingly, hGHi3 did not enhance PGRN-GS expression (see Figure 8 a and 8b), because inclusion of intron 3 slightly lowers PGRN. This suggests that the action of hGHi3 may require an exonic splicing element (ESE) within the wild-type PGRN sequence to exert a cooperative effect on splicing. Use the software ESE finder 3.0 to search for ESE elements in wild-type PGRN and codon-optimized PGRN-GS sequences (see Figure 8 c). The ESE distribution pattern was significantly altered in codon-optimized PGRN-GS compared with wild-type PGRN. Thus, attempts to codon-optimize PGRN by including hGHi3 inadvertently removed the ESE required to provide enhancement.

[0219] Therefore, the intronic enhancer hGHi3 was combined with a PGRN fusion construct carrying...

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Abstract

The present invention relates to the field of recombinant viral vectors suitable for in vivo delivery of therapeutic genes. Described is an adeno-associated virus (AAV) vector comprising (i) a human growth hormone intron 3 (hGHi3) sequence, (ii) a synaptic promoter sequence and / or (iii) a granulin precursor 3'untranslated region (UTR) sequence operatively linked to a polynucleotide sequence encoding a polypeptide of interest. Particular uses of such vectors are to enhance the expression of a polypeptide of interest, such as a granulin precursor (PGRN), to treat subjects having gene mutations or intrinsic polypeptide levels below physiologically normal levels.

Description

[0001] field of invention [0002] The present invention relates to the field of recombinant viral vectors. In particular, the present invention relates to recombinant viral vectors suitable for in vivo delivery of therapeutic genes. [0003] Background of the invention [0004] Frontotemporal dementia (FTD) is the second most common form of dementia in symptomatic persons younger than 65 years. It is characterized by changes in behavior, personality, and language, and can be very distressing for patients, their families, and friends. About 30% of FTD patients have a family history of dementia, and about 20% of them carry a loss of function (LoF) mutation of the GRN gene encoding the protein progranulin (progranulin). Children homozygous for a GRN mutation develop the lysosomal storage disorder neuronal ceroid lipofuscinosis (NCL11), characterized by neurodegeneration and blindness (Rohrer JD et al People, (2015b) Lancet Neurol. 14:253–262). Adults heterozygous for LOF muta...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/11A61K48/00A61P21/00A61P25/00A61P25/14A61P25/16A61P25/28
CPCC12N15/86A61K48/0008A61K48/0058A61K48/0075A61P25/00A61P25/28A61P21/00A61P25/14A61P25/16C12N2750/14143C12N2830/008C12N2800/22C12N2830/42A61K48/005A01K67/0276A01K2217/075A01K2227/105A01K2267/0318
Inventor C·E·D·肖李连福
Owner 伦敦王室学院
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