KR20220018016A - J How to make genetically modified poultry resistant to avian avian leukemia virus - Google Patents

Abstract

The present invention relates to a method for generating a genetically modified poultry having resistance to subgroup J avian leukemia virus, wherein the poultry possesses a W38 deletion introduced in the chNHE1 gene using CRISPR/Cas9-mediated homologous recombination, A native chicken (Gallus gallus domesticus; domestic chicken) or native turkey (Meleagris gallopavo domesticus) that possesses the W38 deletion optionally introduced using CRISPR/Cas9 and specific sgRNA and is resistant to J subgroup avian leukemia virus (ALV) ; is characterized by the fact that domestic turkey) is produced.

Description

J How to make genetically modified poultry resistant to avian avian leukemia virus

The present invention relates to the genetically engineered, permanent and genetically determined resistance of poultry to avian leukemia - a severe poultry disease induced by subgroup J avian leukosis virus.

Avian leukemia is a hematopoietic tumor disease of poultry caused by avian leukemia viruses. The etiology of avian leukemia is associated with avian leukemia viruses (ALVs) of the retroviral family, most commonly with avian leukemia viruses of subgroups A and B, more recently with avian leukemia viruses of subgroup J, and most recently with avian leukemia viruses of subgroup K Associated with a friendly avian leukemia virus. Infection of exogenous ALV is very frequently encountered in poultry populations (Jurajda, V., 2002), but the incidence of clinical disease is generally significantly lower than that of, for example, Mark disease (1 to 1 in infected animals). 5%). Among the forms of leukemia, the relatively most frequent is lymphoid leukosis (LL), and other pathological symptoms of ALV, including osteopetrosis, sarcomas and related tumors, are sporadic or even rare in the field. is found The exception is myelocytosis induced by J subgroup ALV, which began to occur in 1990 in meat-type breeds and broilers in many countries around the world, including the Czech Republic. The economic impact of type J ALV was reflected not only as a direct loss due to death, but also as a negative effect on poultry production (reduced weight gain and decreased egg laying). Moreover, the immunosuppressive effect of potential infection on lymphocytic leukemia exacerbated other infection pathways.

When infecting poultry, the ALV-J virus enters cells through the chNHE1 receptor. The retrovirus is then transcribed by reverse transcription and bound to the nuclear DNA of the host cell as a provirus. The proviral DNA is transcribed into viral RNA, which is translated in the cytoplasm and surrounded by newly formed particles. If the poultry is resistant or not protected by limiting factors, the release of the particles from infected cells leads to the spread of the infection to recipient cells in the individual's body.

In the life cycle of a retrovirus, the sensitivity or resistance to the virus is determined by the presence of an appropriate receptor molecule on the surface of the target cell. The chNHE1 receptor is a cellular glycoprotein with ion exchange functions that appears as a homotrimer. Its first prominent extracellular loop contains amino acid residues important for interaction with ALV-J. Chicken cells lacking this glycoprotein are not receptive to ALV-J infection. Similarly, deletion of amino acid W38 results in resistance to ALV-J but not for all other ALV subgroups. Deletions or substitutions of W38 have been found in NHE1 of pheasants and most coleoptera, and thus susceptible species are native chickens, four wild fowl species, turkeys, and the genus Calipepla, Cole. Includes only species of several New World quails of the genus Colinus and Oreortyx (Placy J, Reinisova M, Kucerova D, Senigl F, Stepanets V, Hron T, Trejbalova K, Elleder) D, Hejnar J.: Identification of New World quails susceptible to infection with avian leukosis virus subgroup J. Journal of Virology 91(3): e02002-16, 2017). According to our study, both native hen breeds and primitive native chicken livestock do not contain alleles with a W38 deletion. Consequently, there is no natural source of genetic diversity to be used to selectively breed resistant animals (Reinisova M, Plachy J, Kucerova D, Senigl F, Vinkler M, Hejnar J.: Genetic Diversity of NHE1, Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species. PloS One 11 (3):e0150589). In countries with advanced poultry industries, systematic and long-term selection of animals suffering from the disease is used to eradicate the disease (eg, in the United States, the last intensive infection of ALV-J disappeared in 2007 (Malhotra S, Justice J 4th, Lee N, Li Y, Zavala G, Ruano M, Morgan R, Beemon K: Complete genome sequence of an American avian leukosis virus subgroup J isolate that causes hemangiomas and myeloid leukosis. Genome Announcements 3(2): pii: e01586-14, 2015)). However, this requires screening of animals that are quite laborious and costly in the breeding process and can only be used in factory farms under full control of the epidemic. In China, where poultry of all ages is commonly sold on the market, where uncontrolled fattening is practiced on small family farms, and virtually throughout Asia and Africa, despite eradication programs launched in recent years, it is difficult to eliminate the disease today. is unrealistic.

The above-mentioned problems are eliminated by the method for generating a genetically modified poultry with resistance to subgroup J avian leukemia virus according to the present invention, which is introduced into the chNHE1 gene using CRISPR/Cas9-mediated homologous recombination. Domestic chickens (Gallus gallus domesticus; domestic chicken) that possess a W38 deletion that has been modified or have a W38 deletion optionally introduced using CRISPR/Cas9 and specific sgRNA, and are resistant to J subgroup avian leukemia virus (ALV) Or it is based on the fact that the native turkey (Meleagris gallopavo domesticus; domestic turkey) is produced.

We generated genetically modified poultry individuals, in which, using gene editing of primordial germ cells (PGC), the tryptophan codon (W38) at position 38 in the chNHE1 ion exchange gene was deleted. Since chNHE1 functions as an ALV-J receptor and W38 is a very important amino acid for its receptor, but not for its ion exchange function, this results in the induction of global resistance to ALV-J. The present invention is based on two previously issued Czech Patents 307102 and 307285.

According to Czech Patent No. 307102, we transfected using 'primordial germ cells' (PGCs), poultry embryonic cells cultured ex vivo, genetically engineered and then implanted into radio-sterilized cockerel testes. It has been found that transgenic individuals can be constituted. The introduced cells restore spermatogenesis, which may result in progeny bearing the same genetic modification as the transplanted PGC. According to Czech Patent No. 307285, we found that deletion of amino acid W38 in the chNHE1 receptor induces global resistance to avian leukemia in an ex vivo model of chicken fibroblasts.

In fact, the method of the present invention corresponds to combining the results of these two above-mentioned Czech patents to create a genetically modified line of native chickens bearing the W38 deletion. By preventing the entry of leukemia viruses into the cells of these individuals, permanent resistance is induced. This protection is genetically determined in these chickens and is therefore transferable to the next generation. Incorporating these resistance-altered individuals into a breeding program transfers this attribute to all individuals, which significantly increases the quality of the breeding flock and ultimately results in poultry meat or egg production. resulting in remarkable economic feasibility. This novel method could have a global impact on poultry health, specifically to native chickens (collectively poultry) and native turkeys.

The method of the present invention for J subgroup avian leukemia is applied to a model of genetically modified individuals with permanent resistance to ALV-J infection made by the present inventors. Thus, it is a pathway to eradicating the disease not only in China and Southeast Asia, but also elsewhere in the world, because the new and more difficult-to-eradicate Chinese ALV- There is a risk of reintroduction of the J strains.

As already mentioned, Czech Patent No. 307285 applies in particular to the susceptibility and resistance of native chickens to ALV-J infection. It constitutes a claim for an isolated DNA molecule or a fragment thereof encoding a mutant protein chNHE1 in which the tryptophan at position 38 is deleted, and additionally an isolated DNA molecule encoding a mutant chNHE1 in which the tryptophan at position 38 is substituted with a glycine or glutamic acid or a claim for a segment thereof. The patent describes a cell-based experimental validation that the NHE1 sequence of native chickens containing the mutation W38 confers total resistance to ALV-J infection! In this patent, the inventors show that when tryptophan in the chNHE1 receptor is substituted with glycine, this substitution induces global resistance to avian leukemia in an ex vivo model of chicken fibroblasts. This fact can only be confirmed/rejected by using CRISPR/Cas9 genome editing to create transformed poultry in which this deletion or substitution has been introduced into its genome. For this purpose, the present inventors used a novel model for the preparation of transgenic animals according to Czech Patent No. 307102, which developed in the testes of sterile recipient roosters after transplantation of donor primordial reproductive chicken cells. It is directly related to the production of avian sperm with high fertilization capacity, thus enabling the production of fully feasible and viable transfected individuals with high efficiencies of about 50%.

Of the accompanying drawings, FIG. 1 shows a diagram of homologous recombination resulting in a W38 deletion in the chicken NHE1 gene. Top is the intron-exon structure of the chNHE1 gene. Exon 1 contains a gRNA target sequence (center) marked with a TGG triplet for W38 (yellow) as a restriction site for Cas9 cleavage (scissors). Synonymous mutation of nucleotides marked in red creates a recognition site for Bsal endonuclease, which then serves for detection of the altered allele. Bottom, sequences of single-stranded oligonucleotides (ssODN) to be used as templates for homologous recombination in chicken and turkey.
FIG. 2 shows a comparison of the sequence (A) of the wild-type chNHE1 allele with the sequence after deletion of the codon for W38 (B). Only those immediately adjacent to W38 indicated by arrows are shown. The sequence is represented as a chromatogram with both a transcribed nucleotide sequence and an amino acid sequence.
3 shows GFP measured by flow cytometry following infection of fibroblasts from embryos of W38 −/- (top left) genotype, W38 +/- (top right) genotype and W38 +/+ (bottom left) genotype. GFP positivity is shown. The x-axis is GFP fluorescence intensity, and the y-axis is cell counts. The percentage of GFP-positive cells is shown in the lower right corner of the histograms. Bottom right, a graph summarizing the percentage of GFP-positive cells among all embryos tested (four for each genotype).
4 shows the quantification results of viremia in chickens of W38 -/-, +/-, and +/+ genotypes (indicated on the x-axis) after infection with the reporter vector RCASBP(J)GFP. is shown Viremia was assessed (on the y-axis) in relative units (fold; fold) of negative controls (infected with non-specific RCAS-A virus) after quantitative RT PCR.

The method of the present invention was conducted by the Institute of Molecular Genetics of the Czech Academy of Sciences, vvi, Prague, CZ, Prague, Czech Republic, and BIOPHARM, Pohori-Chatown, Czech Republic. as, Pohofi-Chotoun, CZ). The following examples of embodiments of the present invention are for illustrative purposes only, and do not limit the present invention in any way.

examples

Example 1

Preparation of native chicken lineages with modified receptor gene chNHE1

Preparation of poultry genetically modified lineages is based on the derivation of primordial germ cells from chicken embryos with an age of 24-96 hours. These cells were cultured and expanded ex vivo from samples of collected embryonic blood, or from the head part of the embryo. The W38 deletion was introduced into the PGC gene using a template for homologous recombination comprising CRISPR/Cas9 having a gRNA specific for the W38 region in the chNHE1 gene and the W38 region ( FIG. 1 ). The construct encoding CRISPR/Cas9 with the appropriate gRNA and the template for homologous recombination were introduced into the PCG by electroporation in the Amaxa system. After electroporation, cells positive for GFP fluorescence associated with CRISPR/Cas9 were selected by sorting of individual cells in a flow cytometer. A well-grown expanded clone with a molecularly identified W38 deletion was then orthotopically transplanted into sterile recipient roosters according to Czech Patent No. 307102. After 5 months, spermatogenesis was observed to be restored and the ejaculate of the recipient cocks was used for artificial insemination of hens, first intramagnally and then vaginal. The offspring consisted exclusively of individuals heterozygous for the W38 deletion. After reaching sexual maturity the heterozygous animals were bred, and in G2 all genotypes, ie, W38 +/+, W38 +/- and W38 +/- and W38 −/-, were segregated in the expected proportions. Consistency with such expected proportions suggests that the W38 deletion does not pose a significant burden on its carrier to reduce its utility within the cultivar. The W38 deletion was performed at all levels using molecular genetic methods, ie, DNA isolation, amplification of a specific fragment of chNHE1 by polymerase chain reaction (PCR), and sequencing of the PCR product {PGC clones, semen of recipient roosters, blood or feather pulp of G1 and G2 individuals}. Exemplary DNA sequences are shown in FIG. 2 .

Example 2

Demonstration of resistance of W38 deletion carriers to ALV-J

Resistance of W38 −/- individuals was demonstrated by three independent methods routine in the field of virology research and which have been established specifically for ALV-J.

A. Embryonic fibroblasts ex vivo infection

After the embryos of G2 individuals were incubated until day 10 of development, a culture of embryonic fibroblasts was prepared. The W38 genotype was verified by DNA isolation, PCR and sequencing. Cultures of embryonic fibroblasts were infected with a virus having ALV-J receptor specificity, in particular, the reporter vector RCASBP(J)GFP. For easy virus detection, the reporter vector contained green fluorescent protein; A reporter gene for GFP) is transduced. Quantitative evaluation of GFP was made using flow cytometry. Embryonic fibroblasts with the W38+/+ genotype and W38+/- genotype showed the same GFP positivity in about 90% of the cells, indicating almost complete virus spread. In contrast, embryonic fibroblasts with the W38-/- genotype revealed GFP positivity in less than 0.05% of cells, which corresponds to the natural background given by autofluorescence ( FIG. 3 ). It can be concluded that the W38-/- genotype has complete resistance to ALV-J infection at the level of embryonic fibroblasts.

B. Chickens in vivo infection

Chickens of the W38 +/+, W38 +/- and W38 +/- and W38 −/- genotypes were infected with the reported vector RCASBP(J)GFP at the age of several days to 2 months, which retains ALV-J receptor specificity, eg, HPRS103 It is more aggressive than the ALV-J of the same prototype strain. The normal course of ALV-J infection manifests itself as transient viremia, which occurs after a few days depending on the age of the infected individual, and typically peaks within 10 days, after which it peaks 1 to 2 Lasts for a week. The in vivo presence of the virus is determined by the quantitative detection of viral genomic RNA (viral genomic RNA) using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), molecularly, and replication. A replication-competent introduced virus complements the defective virus, which is then based on the number of focus-forming transformed cells that form the lesion. Secondary quantification was validated at this time in both methods, biologically by a complementation assay. The second method of detection verifies the presence of a biologically active virus as well as RNA. In both cases, the substances tested corresponded to the sera of infected animals collected at two time points, 1 week post-infection and 2 weeks post-infection. All chickens of the W38-/- genotype (a total of 5 chickens) were tested negative at both time points using RT-qPCR. The W38 +/+ genotype and W38 +/- genotype (10 chickens total) were positive at later time points with one exception; Three chickens were negative at the first collection time point, indicating a slow development of viremia during the first week and an increase in viremia during the second week. The only negative case here may refer to an unsuccessful infection (failure of infection), for example due to a defective inoculation. These results are summarized in FIG. 4 . In this complementarity assay, sporadic positive cases at the first collection point were found only for the W38 +/+ genotype and the W38 +/- genotype. At the second time point, samples of animals with these genotypes were positive with one exception (the same individual as in the previous experiment), whereas all W38 −/- genotypes remained negative (Table 1). In summary, we can conclude that deletion W38 induces complete resistance to the virus when ALV-J is inoculated into the circulating blood of juvenile individuals. This type of infection best simulates infection under domestic conditions.

Table 1 shows the validation of viremia in W38 −/- genotype, +/- genotype and +/+ genotype (control wild-type genotype) chickens after infection with the report vector RCASBP(J)GFP. Viremia was characterized by a titre of complemented defective virus 16Q and terminal dilution in two serum samples, 6 and 13 days after infection. post infection).

C. Tumor induction by ALV-J-pseudotyped virus

To simulate the formation of solid tumors after ALV-J infection, we tested a transgenic virus present in quail cell line 16Q and containing the -src oncogene by a virus with ALV-J receptor specificity, i.e., It was pseudotyped by the RCASBP(J)GFP vector. This results in an acutely transforming virus with the oncogene v-src surrounded by the ALV-J virus glycoprotein, which in our case is a rapidly growing sarcomas at the site of inoculation in the wing web. ) has the ability to induce We inoculated chickens between 10 days and 2 months of age, and the onset and growth of sarcomas was monitored for one month. All chickens of genotype W38 +/+ and genotype W38 +/- genotypes developed progressively growing sarcomas, whereas chickens of genotype W38 developed no tumors in any case. Tumors in animals of the W38 +/- genotype grew faster than those in chickens of the W38 +/- genotype, indicating a mild negatively dominant effect of the allele carrying the W38 deletion. This is probably related to chNHE1 trimerization. It can be concluded that this experiment demonstrated (complete) total resistance to ALV-J in the case of a homozygous W38 deletion.

Examples mentioned above (recently Koslova A, Trefil P, Mucksova J, Reinisova M, Plachy J, Kalina J, Kucerova D, Geryk J, Krchlikova V, Lejckova B, Hejnar J: Precise CRISPR/Cas9 Editing of the NHE1 Gene Renders Chickens Resistant to the J Subgroup of Avian Leukosis Virus. Proc Natl Acad Sci US A. 117::2108-2112, published in full as 2020) is a homozygous deletion in the chNHE1 gene, made using the method of the present invention. A new line of native chickens harboring W38 preserves normal growth properties, reproductive properties, and useful properties and exhibits excellent economic effects without any apparent adverse effects while being completely resistant to the pathogenic virus ALV-J. Document it conclusively.

This novel method of producing genetically modified poultry with resistance to sub-J avian leukemia virus provides a novel and novel solution for eradication of type J avian leukemia ALV, resulting in permanent resistance to ALV-J infection. We present a newly created model of genetically modified individuals with The method therefore represents a completely revolutionary method of eradicating the disease not only in poultry husbandry powers such as China and Southeast Asia, but also elsewhere in the world, since the disease is re-introduced in regions where its etiology has already been eradicated. This is because there is a potential risk of becoming

References:

JURAJDA, Vladimir. Nemoci drubeze a ptactva - virove infection. 1. vyd. Brno: Edicni stfedisko VFU. Brno, 2002. 184 s. ISBN 80-7305-436-1.

Malhotra S, Justice J 4th, Lee N, Li Y, Zavala G, Ruano M, Morgan R, Beemon K: Complete genome sequence of an american avian leukosis virus subgroup j isolate that causes hemangiomas and myeloid leukosis. Genome Announcements 3(2): pii: e01586-14, 2015

Plachy J, Reinisova M, Kucerova D, Senigl F, Stepanets V, Hron T, Trejbalova K, Elleder D, Hejnar J.: Identification of New World quails susceptible to infection with avian leukosis virus subgroup J. Journal of Virology 91(3) : e02002-16, 2017

Reinisova M, Plachy J, Kucerova D, Senigl F, Vinkler M, Hejnar J.: Genetic Diversity of NHE1 , Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species. PloS One 11(3):e0150589), 2016

Koslova A, Trefil P, Mucksova J, Reinisova M, Plachy J, Kalina J, Kucerova D, Geryk J, Krchlikova V, Lejckova B, Hejnar J: Precise CRISPR/Cas9 Editing of the NHE1 Gene Renders Chickens Resistant to the J Subgroup of Avian Leukosis Virus. Proc Natl Acad Sci U S A. 117::2108-2112, 2020

Czech Patent No. 307102

Czech Patent No. 307285

Claims (1)

A method for making a genetically modified poultry resistant to J subgroup avian leukemia virus, the method comprising:
This poultry harbors a W38 deletion introduced in the chNHE1 gene using CRISPR/Cas9-mediated homologous recombination, or optionally a W38 deletion introduced using CRISPR/Cas9 and specific sgRNA, resulting in a J subgroup avian leukemia virus. A method, characterized by the fact that domestic chickens (Gallus gallus domesticus; domestic chicken) or native turkeys (Meleagris gallopavo domesticus; domestic turkey) with resistance to (ALV) are produced.

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