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Application of lentiviral vector for expressing factor VIII

A technology of lentiviral vectors and nucleic acid molecules, which is applied in the field of lentiviral vectors expressing factor VIII, and can solve problems such as inconvenient cost and high cost of FVIII protein

Pending Publication Date: 2020-11-10
比奥维拉迪维治疗股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Giving so frequently is inconvenient and costly
[0007] The major obstacle to delivering low-cost recombinant FVIII protein to patients is the high cost of commercial production

Method used

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  • Application of lentiviral vector for expressing factor VIII
  • Application of lentiviral vector for expressing factor VIII
  • Application of lentiviral vector for expressing factor VIII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0526] Example 1. Codon optimization strategy

[0527] Eight codon-optimized BDD FVIII variants including coFVIII-3 (SEQ ID NO: 1; Figure 1A ), coFVIII-4 (SEQ ID NO: 2; Figure 1B ), coFVIII-5 (SEQ ID NO: 70; Figure 1C ), coFVIII-6 (SEQ ID NO:71; Figure 1D ), coFVIII-52 (SEQ ID NO: 3; Figure 1E ), coFVIII-62 (SEQ ID NO: 4; Figure 1F ), coFVIII-25 (SEQ ID NO: 5; Figure 1G ), and coFVIII-26 (SEQ ID NO:6; Figure 1H ). Codon optimization was facilitated using the online tool Eugene and several codon usage parameters were monitored as previously described (see Gaspar et al., "EuGene: maximizing synthetic gene design for heterologous expression," Bioinformatics 28:2683-84 (2012)) Such as codon adaptation index (CAI) and relative synonymous codon usage (RSCU) (Table 5). All variants were adjusted to have a CAI > 83% and an RSCU > 1.63, while the parental B-domain deleted FVIII sequence had a CAI of 74% and an RSCU of 1.12 before optimization (Table 5).

[0528] Table ...

Embodiment 2

[0534] Example 2. Cloning and expression of coFVIII variants from the pcDNA3 plasmid

[0535] Expression plasmids containing various FVIII variants were designed for in vivo expression. Convert the unoptimized BDD FVIII ( Figure 1I ; SEQ ID NO: 16) and coFVIII-1 ( Figure 11Z ; SEQ ID NO:68) polynucleotide was cloned into the pcDNA3 backbone (Invitrogen), wherein the CMV promoter was replaced with the ET promoter (see image 3 ). The resulting plasmids FVIII-311 (BDDFVIII) and FVIII-303 (coFVIII-1 ) drive the expression of unoptimized BDD FVIII and coFVIII-1, respectively.

[0536] In vivo expression of FVIII-311 and FVIII-303 was evaluated in Hem A mice by hydrodynamic injection of 5 μg DNA / mouse of FVIII-303 or FVIII-311. Plasma samples were collected at 24, 48 and 72 hours post-injection, and FVIII activity was determined by a FVIII-specific chromogenic assay.

[0537] like Figure 4 As shown in , at 72 hours after injection, the plasma FVIII activity (BDDFVIII; squa...

Embodiment 3

[0538] Example 3. Cloning and expression of coFVIII variants using a lentiviral vector system

[0539] To further assess the expression levels of codon-optimized BDD FVIII variants, the coding sequences were cloned into lentiviral plasmids under the control of the ET promoter (see Amendola et al., "Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters, "Nature Biol. 23:108-16 (2005); International Publication No. WO 2000 / 066759 Al). The plasmid map of pLV-coFVIII-52 is shown in Figure 5 and in the same way to construct BDD containing unoptimized FVIII (LV-2116), coFVIII-1 (LV-coFVIII-1), coFVIII-3 (LV-coFVIII-3), coFVIII-4 (LV-coFVIII- 4), coFVIII-5 (LV-coFVIII-5), and coFVIII-6 (LV-coFVIII-6), coFVIII-62 (LV-coFVIII-62), coFVIII-25 (LV-coFVIII-25), and coFVIII -26 (LV-coFVIII-26), but the coFVIII-52 fragment was replaced by the indicated coding sequence using each of the NheI and SalI sites (Table 6).

[0540] Table 6: Expressi...

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Abstract

The present disclosure provides lentiviral vectors comprising codon optimized Factor VIII sequences, and methods of using such lentiviral vectors. The liver-targeted lentiviral vectors disclosed herein can be used for gene therapy, wherein the lentiviral gene delivery enables stable integration of the transgene expression cassette into the genome of targeted cells (e.g., hepatocytes) of pediatric(e.g., neonatal) or adult subjects, achieving an improvement in FVIII expression (for example, a 100-fold improvement) at low lentiviral vector doses (e.g., 5x1010 or lower, such as 1.5x109 or lower,or 1x108 TU / kg or lower). The present disclosure also provides methods of treating bleeding disorders such as hemophilia (e.g., hemophilia A) comprising administering to a subject in need thereof a liver-targeted lentiviral vector comprising a codon optimized Factor VIII nucleic acid sequence at low dosages (1*108 TU / kg or lower to 1.5*1010 TU / kg).

Description

[0001] related application [0002] This application claims priority to U.S. Provisional Patent Applications Serial No. 62 / 625,145 filed February 1, 2018, Serial No. 62 / 671,915 filed May 15, 2018, and Serial No. 62 / 793,158 filed January 16, 2019 right, the entire disclosure of said application is hereby incorporated by reference. [0003] References to Electronically Submitted Sequence Listings [0004] The Sequence Listing, filed electronically as an ASCII text file (name: 609628_SA9_460PC_Sequence_Listing.txt; size: 204,203 bytes; and date created: January 31, 2019), is hereby incorporated by reference in its entirety. Background technique [0005] The blood coagulation pathway involves in part the formation of an enzyme complex of Factor VIIIa (FVIIIa) and Factor IXa (FIXa) (X enzyme complex) on the surface of platelets. FIXa is a serine protease whose catalytic activity is relatively weak without its cofactor FVIIIa. The X enzyme complex cleaves Factor X (FX) to Factor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/867C07K14/755
CPCA61K48/0058A61K48/0066C07K14/755C12N15/86C07K2319/00C07K2319/02C07K2319/31C12N2740/16043C12N2800/22C12N2830/008C12N2830/48A61K39/12A61P7/00A61K38/37A61K48/0075
Inventor A·安诺尼A·康托尔D·德拉格刘彤瑶M·米拉尼J·莫菲特L·纳迪尼S·帕塔罗约-怀特R·彼得斯A·塞雷金
Owner 比奥维拉迪维治疗股份有限公司