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Donor pig for xenotransplantation

A technique of xenotransplantation, donor pig, application of tissue and/or cells to avoid xenograft rejection, selection of pigs lacking infectious porcine endogenous retroviruses, enhancement of donor organs, capable of addressing genetic damage to animals Consequences and other issues

Pending Publication Date: 2022-03-25
NZENO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] However, the combination of deactivating / removing all PERV and xenoantigen genes using gene editing techniques (eGenesis talks about introducing "double-bit" gene editing to reduce the possibility of organ rejection (Weintraub, 2019)) could mean that the resulting cloned animals have tens of gene editing, which could harm animals and / or have unforeseen genetic consequences

Method used

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  • Donor pig for xenotransplantation
  • Donor pig for xenotransplantation
  • Donor pig for xenotransplantation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0169] This example describes a method for the analysis of PERV content in wild boar by whole genome sequencing.

[0170] 1.1 Method

[0171] Wild boar male 1 is a male AI pig known as pathogen free (DPF) and was identified as PERV-C negative by PCR-based testing.

[0172] Complete genome sequencing of wild boar male 1 was performed by Macrogen Inc. under contract. Binary Alignment Map (BAM) files (showing filtered DNA sequence reads mapped to a reference genome with duplicate reads removed) and variant calling format (VCF) files (shown by analysis) were obtained from Macrogen Inc. DNA sequence variants identified from information obtained from aligned reads) and used for further analysis. Regions without coverage from the consensus sequence were replaced with the letter "N" using BCF tools (BCFtools). Geneious was used to BLAST the wild boar male 1 genome to the entire PERV-C sequence of KC116219 (8678bp; NCBI reference sequence).

[0173] 1.2 Results

[0174] 1.2.1 Geno...

Embodiment 2

[0197] This example describes another example of analysis of PERV content in wild boar by whole genome sequencing.

[0198] 2.1 Method

[0199] Wild boar male 2 is an AI male pig known as pathogen free (DPF) and was identified as PERV-C positive by PCR-based testing. Whole-genome sequencing of wild boar male 2 was performed as described in Example 1, Section 1.2.

[0200] 2.2 Results

[0201] 2.2.1 Genome sequencing and identification of PERV sequences

[0202] A total of 25 queries covering more than 50% of the genomic region were detected (KC116219)( Figure 8 ), only 8 covered more than 90%. These were selected as candidates for potential functional PERVs for further analysis (Table 3).

[0203] Table 3. Genomic regions of wild boar male 2 covering more than 90% of the PERV-C sequence (KC116219).

[0204]

[0205] The 8 fragments were combined with variants A, B and C (variant C: KC116219, KC116221, AM229311; recombinant variant A / C: AY570980, AY953542; variant A: AJ...

Embodiment 3

[0221] This example describes the generation and characterization of the NZK1 cell line derived from Auckland Island (AI) porcine kidney.

[0222] 3.1 Method

[0223] 3.1.1 Isolation of kidney-derived AI porcine cells

[0224] Kidney-derived primary AI porcine cells were isolated from tissue samples from two individual animals. One was a male neonatal piglet (#18 / PA007) that died unexpectedly shortly after birth on February 6, 2018, and the resulting cell line was denoted NZK1.

[0225] Kidney cells were isolated based on the method from Richter et al. (2012). Briefly, 1 cm from the renal cortex and medulla were first washed 3 Cube the tissue, then centrifuge after mincing. Afterwards, the tissue was resuspended in 15 ml of Hank's buffered saline solution containing 0.1% (w / v) collagenase (type I or II; Invitrogen) and incubated at 37°C with stirring for 1 to 1.5 h. The supernatant containing kidney cells was filtered through a 100 μm mesh, washed with Dulbecco's modified...

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Abstract

The invention relates to a new strain of pigs very suitable for xenotransplantation. The first new porcine line lacks a functional porcine endogenous retrovirus and thus is suitable as a donor for tissue and / or cell xenotransplantation into a human recipient. These pigs can also be used as base pigs for further operation, e.g., by gene editing of heteroantigens to produce a second new strain of pigs that is not only free of infectious porcine retroviruses, but also free of major heteroantigens that cause super-acute organ rejection. These pigs can be used to transplant the entire organ, tissue and / or cell into a human recipient. The invention also relates to a method of selecting a pig lacking an infectious porcine endogenous retrovirus, as well as their use for tissue and / or cell xenotransplantation into a human, as well as a method of gene editing of xenoantigens of selected pigs to further enhance donor organs, tissues and / or cells to avoid xenotransplantation rejection.

Description

technical field [0001] The present invention generally relates to new strains of pigs well suited for xenotransplantation. The first new porcine strain lacks a functional porcine endogenous retrovirus and is therefore suitable as a donor for tissue and / or cell xenotransplantation into human recipients. These pigs can also be used as base pigs for further manipulation, such as by gene editing of xenoantigens to generate a second novel strain of pigs that is not only free of infectious porcine retroviruses, but also free of the major pathogens that cause hyperacute organ rejection. Xenoantigen. These pigs can be used to transplant whole organs, tissues and / or cells into human recipients. [0002] The invention also relates to methods of selecting pigs lacking infectious porcine endogenous retroviruses, and their use for xenotransplantation of tissues and / or cells into humans, and methods of gene editing of xenoantigens of selected pigs, to Methods of further enhancing donor o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/02A01K67/027C12N15/877C12Q1/6888C12Q1/70
CPCA01K67/0276C12N2517/04C12N2501/01C12N15/8778C12N15/8509C12Q2600/124A01K2217/077A01K2267/025A01K2217/15C12Q1/6888A01K2217/056A01K67/0278C12Q1/702A01K2227/108A01K2207/15C12N15/907C12Q1/6881C12Q1/6883
Inventor P·L·J·谭O·加尔卡文科R·B·埃利奥特
Owner NZENO LTD
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