Mirna silencing of CD163 in pigs

Abstract

The present disclosure relates to methods of creating Porcine Reproductive and Respiratory Syndrome virus (PRRSv) resistant pigs by gene silencing. In particular, the methods comprise using tools such as CRISPR / CAS9 to edit genes that either directly encode or encode molecules that are processed into non-coding RNA molecules to induce or modify their silencing activity against CD163 and / or in cells of pigs for the purpose of promoting PRRSv resistance.

Description

Atty. Dkt. No. 102726-0300(TD-20-2024-W01)MIRNA SILENCING OF CD163 IN PIGSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63,721,346, filed November 15, 2024, the content of which is incorporated herein by reference in its entirety for any and all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 07, 2025, is named 102726-0300_SL.xml and is 211,122 bytes in size.TECHNICAL FIELD

[0003] The present technology relates generally to methods for improving traits or fitness in pigs via gene editing. In particular, the methods comprise modifying genes that encode / are processed into non-coding RNA molecules to induce or modify their silencing activity against CD 163 in pigs to provide PRRS virus resistance.BACKGROUND

[0004] The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art.

[0005] Viral infections are a major source of morbidity and mortality in the livestock industry. In particular, Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSv). PRRS is the most economically important disease of domestic swine in North America, Europe, and Asia, costing producers in North America more than $600 million annually.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0006] Currently, there are no effective treatment programs for acute PRRS. As a result, when incidence of PRRS is detected on a farm, depopulation, sufficient cleaning / disinfection, and proper disposal of the carcasses must be used to eliminate the virus. In more extreme cases, whole herd depopulation-repopulation has been documented as an effective method of eliminating the PRRSv from endemically infected herds; however, this method results in significant loss.

[0007] Vaccines for PRRSv do exist; however, these vaccines have been unable to control the disease largely due to the genetic diversity. Consequently, prevention of infection is currently the best control measure. As a prophylactic measure, farms in a country or zone where PRRSv exists must use stringent control measures, involving an assessment of the health status of replacement gilts and boars, as well as 45-60 days of isolation and acclimatization for incoming stock.

[0008] In recent years, more attention has been given to the role CD 163 may play in the occurrence of PRRS. Despite the significant heterogeneity in strains of PRRSv, strains of PRRSv all share a tropism for CD 163 -positive cells. Although CD163 is a virus receptor, the CD 163 scavenger receptor is also involved in the adhesion of monocytes to endothelial cells. Functions and a detailed description of CD163 are provided in Onofre, G., et al., ACTAMEDICA, 2009, 52, 57-61.

[0009] CD163 is a 130 kDa type 1 membrane protein considered to be a fusion receptor for the PRRS virus; it is mapped to chromosome 5 in pigs. The basic transcript encodes for a protein of 1076 amino acids. There are five reported isoforms of CD 163; three of the isoforms display different splicing forms of their cytoplasmic domains. Generally, however, the genomic molecular sequence of CD 163 comprises 17 exons coding for a peptide signal sequence, nine scavenger receptor cysteine-rich (SRCR) domains, two proline serine threonine (PST) linker domains, a cytoplasmic domain, and a short cytoplasmic tail. CD163 has been described as the receptor for PRRSv. Domain 5 (SRCR5) of the protein is the interaction site for the virus. Exon 7 of CD 163 encodes the SRCR domain 5 (SRCR5) that serves as an interaction site for the PRRSv in vitro. Burkard (Burkard, C., et al., PLoS Pathog., 2017, 23, 13, el 006206.) demonstrated that removal of CD 163 exon 7 confersAtty. Dkt. No. 102726-0300(TD-20-2024-W01)PRRSv resistance to porcine macrophages. The guides used in that work, however, may lack sufficient activity and specificity for gene editing as part of a commercial breeding program. Further work by Whitworth and colleagues included creating a 123 bp deletion in Exon 7 (Whitworth, K.M., etal., Biol. Reprod., 2014, 91, 1-13). Whitworth etal. (Whitworth, K.M., etal., Nature Biotechnology, 2016, 34, 20-22) reported the preparation of PRRSv resistant pigs by knocking out the function of CD 163. This genomic approach must be carefully targeted to avoid knocking out critical gene functions while avoiding infection by the PRRSv.

[0010] Genome editing includes altering the genome by deleting, inserting, or substituting specific nucleic acid sequences. The alteration can be gene- or location-specific. Genome editing can use site-directed nucleases, such as Cas proteins and their cognate polynucleotides.

[0011] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute the CRISPR-Cas system.

[0012] Cas9 is an exemplary Type II CRISPR Cas protein. Cas9 is an endonuclease that can be programmed by the tracrRNA / crRNA to cleave, in a site-specific manner, a DNA target sequence using two distinct endonuclease domains (HNH and RuvC / RNase H-like domains) (see U.S. Patent Application Publication No. 2014-0068797, published 6 Mar. 2014; see also Jinek, M., et al., Science, 2012, 337, 816-821 and Karvelis, T, et al., Genome Biology, 2015, 16, 253).

[0013] There is a need to improve the health of a porcine herd by editing the CD 163 gene using guides for improved editing activity and reduced unintended edits, while conferring resistance to PRRSv.

[0014] Two of the most powerful genetic therapeutic technologies developed thus far are gene therapy, which enables restoration of missing gene function by viral transgene expression, and RNA interference (RNAi), which mediates repression of defective genes by knockdown of the target mRNA.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0015] Despite promise and recent success, gene therapy and RNAi have limitations that preclude their utility for a large number of indications. For example, viral gene therapy may cause mutagenesis at the integration site and result in dysregulated transgene expression (Howe, S.J., etal., J. Clin. Invest., 2008, 118, 3143-3150). Meanwhile, the use of RNAi is limited to targets for which gene knockdown is beneficial. Also, RNAi often cannot fully repress gene expression due to the transient nature of the delivered siRNA and the lack of silencing amplification mechanisms like in plants or nematodes, and is therefore unlikely to provide a benefit in which complete repression of gene function is necessary for efficacy. The current main obstacle of RNA-based interventions is efficient and effective RNA delivery into cells. Although some delivery agents can enhance therapeutic RNA endocytosis, only a very small fraction, less than 0.01, escapes from the endosomes and are biologically active (Dowdy, S.F., Nature Biotechnol., 2017, 35, 222-229).

[0016] The present inventors have developed a novel, RNAi-like approach to preventing or reducing PRRSv infection in pigs.SUMMARY

[0017] In one aspect, the present disclosure provides a porcine animal or porcine cell comprising one or more gene-edited chromosomal sequences, each encoding a non-coding RNA molecule that silences an endogenous CD 163 gene. In some embodiments, the noncoding RNA molecule encodes or is processed into an siRNA having a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 51-95. In some embodiments, the noncoding RNA molecule decreases the expression or activity of CD 163 protein as compared to the expression or activity in a porcine animal or porcine cell that lacks the one or more gene-edited chromosomal sequences. In some embodiments, the porcine animal or porcine cell is resistant to Porcine Reproductive and Respiratory Syndrome virus (PRRSv). In some embodiments, the one or more gene-edited chromosomal sequences encoding the non-coding RNA molecule reduces susceptibility of the porcine animal or porcine cell to infection by PRRSv. In some embodiments, the one or more gene-edited chromosomal sequences comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-50. In some embodiments, the non-coding RNA molecule is only expressed in pulmonaryAtty. Dkt. No. 102726-0300(TD-20-2024-W01)alveolar macrophages. In some embodiments, the one or more gene edited-chromosomal sequences comprise an edit of an endogenous non-coding RNA molecule that redirects silencing activity of the encoded non-coding RNA molecule to CD 163. In some embodiments, the non-coding RNA molecule encodes or is processed into an siRNA having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 51-95. In some embodiments, the one or more gene-edited chromosomal sequences comprise a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-50. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 8; (b) SEQ ID NO: 9; (c) SEQ ID NO: 10; (d) SEQ ID NO: 40; (e) SEQ ID NOs: 8 and 10; (f) SEQ ID NOs: 9 and 10; (g) SEQ ID NOs: 9 and 40; and (h) SEQ ID NOs: 10 and 40. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 8; (b) SEQ ID NO: 9; (c) SEQ ID NO: 40; (d) SEQ ID NOs: 8 and 10; (e) SEQ ID NOs: 9 and 40; and (f) SEQ ID NOs: 10 and 40. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 8; (b) SEQ ID NO: 9; (c) SEQ ID NO: 10; (d) SEQ ID NOs: 9 and 10; (e) SEQ ID NOs: 9 and 40; and (f) SEQ ID NOs: 10 and 40. In some embodiments, the porcine animal is a gene-edited pig. In some embodiments, the present disclosure provides an isolated porcine cell obtained from the porcine animal comprising the one or more gene-edited chromosomal sequences. In some embodiments, the cell is selected from a germ cell, a somatic cell, a primary cell, a stem cell, an embryonic stem cell, an adult stem cell, a hematopoietic stem cell, a mesenchymal stem cell, a gamete cell, a zygote cell, a blastocyst cell, an embryo, or a fetus. In some embodiments, the germ cell is a spermatogonia or an oogonia. In some embodiments, the gamete cell is a sperm cell or an oocyte.

[0018] In another aspect, the present disclosure provides an siRNA comprising any one of SEQ ID NOs: 51-95.

[0019] In another aspect, the present disclosure provides an miRNA comprising any one of SEQ ID NOs: 1-50.

[0020] In another aspect, the present disclosure provides a method for producing a porcine animal or porcine cell comprising one or more gene-edited chromosomal sequences, eachAtty. Dkt. No. 102726-0300(TD-20-2024-W01)encoding a non-coding RNA molecule that silences an endogenous CD 163 gene, the method comprising contacting the porcine animal or porcine cell with an effective amount of a DNA editing agent, wherein the DNA editing agent is configured to edit a chromosomal sequence to produce one or more gene-edited chromosomal sequences, each encoding a non-coding RNA molecule that silences an endogenous CD 163 gene, thereby producing a porcine animal or porcine cell having decreased expression or activity of CD 163 protein as compared to the expression or activity of CD 163 in a porcine animal or porcine cell that lacks the edited chromosomal sequence. In some embodiments, the DNA editing agent comprises at least one guide RNA (gRNA) operably linked to a constitutive or inducible promoter. In some embodiments, the DNA editing agent comprises an endonuclease. In some embodiments, the DNA editing agent comprises a meganuclease, a zinc finger nuclease, a transcriptionactivator like effector nuclease, or CRISPR. In some embodiments, the non-coding RNA molecule is produced via homologous DNA repair (HDR) with a donor template sequence comprising a modification of interest. In some embodiments, the non-coding RNA molecule encodes or is processed into a siRNA. In some embodiments, the siRNA is selected from the group consisting of: SEQ ID NOs: 51-95. In some embodiments, the one or more gene-edited chromosomal sequences comprise a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-50. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 8; (b) SEQ ID NO: 9; (c) SEQ ID NO: 10; (d) SEQ ID NO: 40; (e) SEQ ID NOs: 8 and 10; (f) SEQ ID NOs: 9 and 10; (g) SEQ ID NOs: 9 and 40; and (h) SEQ ID NOs: 10 and 40. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 81; (b) SEQ ID NO: 9; (c) SEQ ID NO: 40; (d) SEQ ID NOs: 8 and 10; (e) SEQ ID NOs: 9 and 40; and (f) SEQ ID NOs: 10 and 40. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 8; (b) SEQ ID NO: 9; (c) SEQ ID NO: 10; (d) SEQ ID NOs: 9 and 10; (e) SEQ ID NOs: 9 and 40; and (f) SEQ ID NOs: 10 and 40.

[0021] In another aspect, the present disclosure provides, a method for producing a porcine animal or porcine cell resistant to Porcine Reproductive and Respiratory Syndrome virus (PRRSv), the method comprising contacting the porcine animal or porcine cell with an effective amount of a DNA editing agent, wherein the DNA editing agent is configured to editAtty. Dkt. No. 102726-0300(TD-20-2024-W01)a chromosomal sequence to produce one or more gene-edited chromosomal sequences, each encoding a non-coding RNA molecule that silences an endogenous CD 163 gene, thereby producing a porcine animal or porcine cell having decreased expression or activity of CD 163 protein as compared to the expression or activity of CD 163 in a porcine animal or porcine cell that lacks the edited chromosomal sequence. In some embodiments, the porcine animal or porcine cell is infected with PRRSv, the animal or cell has an increased resistance to PRRSv infectivity as compared to an infected porcine animal or porcine cell that lacks the one or more gene-edited chromosomal sequences. In some embodiments, the DNA editing agent comprises at least one guide RNA (gRNA) operably linked to a constitutive or inducible promoter. In some embodiments, the DNA editing agent comprises an endonuclease. In some embodiments, the DNA editing agent comprises a meganuclease, a zinc finger nuclease, a transcription-activator like effector nuclease, or CRISPR. In some embodiments, the non-coding RNA molecule is produced via homologous DNA repair (HDR) with a donor template sequence comprising a modification of interest. In some embodiments, the non-coding RNA molecule encodes or is processed into a siRNA. In some embodiments, the siRNA is selected from the group consisting of: SEQ ID NOs: 51-95. In some embodiments, the one or more gene-edited chromosomal sequences comprise a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-50. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 8; (b) SEQ ID NO: 9; (c) SEQ ID NO: 10; (d) SEQ ID NO: 40; (e) SEQ ID NOs: 8 and 10; (f) SEQ ID NOs: 9 and 10; (g) SEQ ID NOs: 9 and 40; and (h) SEQ ID NOs: 10 and 40. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 8; (b) SEQ ID NO: 9; (c) SEQ ID NO: 40; (d) SEQ ID NOs: 8 and 10; (e) SEQ ID NOs: 9 and 40; and (f) SEQ ID NOs: 10 and 40. In some embodiments, the one or more gene-edited chromosomal sequences are selected from the group consisting of: (a) SEQ ID NO: 8; (b) SEQ ID NO: 9; (c) SEQ ID NO: 10; (d) SEQ ID NOs: 9 and 10; (e) SEQ ID NOs: 9 and 40; and (f) SEQ ID NOs: 10 and 40.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)DETAILED DESCRIPTION

[0022] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3d edition; the series Ausubel, etal. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N. Y.);MacPherson, et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson, et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. ( \999 Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg, et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0023] In general, the present technology relates to using the Genome Editing induced Gene Silencing (GEiGS® solutions; Tropic Biosciences, Norwich, United Kingdom) platform to redirect endogenous non-coding RNA molecules to silence expression of the CD 163 gene in pigs.

[0024] Recent advances in genome editing techniques have made it possible to alter DNA sequences in living cells by editing a few nucleotides in mammalian cells by creating a sitespecific DNA double-strand break (DSB) in the genome and then allowing the cell'sAtty. Dkt. No. 102726-0300(TD-20-2024-W01)endogenous DSB repair machinery to fix the break (such as by non-homologous end-joining (NHEJ) or homologous recombination (HR)), in which the latter can allow precise nucleotide changes to be made to the DNA sequence (Porteus, M., Annu. Rev. Pharmacol. Toxicol.. , 2016, 56, 163- 190). This gene editing technology allows scientists to make precise changes in the genome to utilize various nucleic acid functions.

[0025] The GEiGS® platform, previously disclosed in WO 2019 / 058253 and WO2020 / 183419, the contents of which are incorporated herein in their entirety, utilizes a eukaryotic cell's endogenous non-coding RNA molecules including e.g., RNA silencing molecules (e.g., siRNA, miRNA, piRNA, tasiRNA, antisense RNA, etc.) and modifies them to target any RNA target of interest. Using GEiGS®, the present disclosure enables screening of potential non-coding RNA molecules, editing several nucleotides in these endogenous RNA molecules, and redirecting their activity and / or specificity to effectively and specifically target any RNA of interest, in this instance the porcine CD 163 gene. The nucleotide sequence of the redirected endogenous RNA molecule is referred to as a GEiGS ® solution. The gene editing technology described herein does not necessitate the classical molecular genetic and transgenic tools comprising expression cassettes that have a promoter, terminator, and selection marker. Moreover, the gene editing technology disclosed herein comprises genome editing of a non-coding RNA molecule (e.g., endogenous) that is stable and heritable.Definitions

[0026] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the technology are described below in various levels of detail in order to provide a substantial understanding of the present disclosure. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0027] Throughout this application, various embodiments of the present technology may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitationAtty. Dkt. No. 102726-0300(TD-20-2024-W01)on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0028] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0029] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0030] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0031] As used herein, the term “effective amount” refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. In some embodiments, an effective amount of a modified gene encoding / processed into a non-coding RNA molecule (e.g., a silencing-dysfunctional miRNA molecule) provided herein, may refer toAtty. Dkt. No. 102726-0300(TD-20-2024-W01)the amount of the modified gene encoding / processed into a non-coding RNA molecule that is sufficient to induce editing of a target RNA site specifically recognized by the modified non-coding RNA molecule. As will be appreciated by the skilled artisan, the effective amount of an agent may vary depending on various factors such as, for example, the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.

[0032] As used herein, the term “redirects a silencing specificity” refers to reprogramming the original specificity of the non-coding RNA (e.g., RNA silencing molecule) towards a non-natural target of the non-coding RNA (e.g., RNA silencing molecule). Accordingly, the original specificity of the non-coding RNA is destroyed ( / .< ., loss of function) and the new specificity is towards an RNA of interest instead of the natural target. It will be appreciated that the designed non-coding RNA molecule of some embodiments of the present technology can have some off-target specificity effect(s) provided that it does not affect the growth, differentiation, or function of the eukaryotic cell or organism.

[0033] The term “RNA silencing,” “silencing,” “gene silencing,” “silenced,” “silenced RNA,” “silenced gene,” “silence,” or RNAi refers to a cellular regulatory mechanism in which non-coding RNA molecules (the “RNA silencing molecule” or “RNAi molecule”) mediate, in a sequence specific manner, co- or post-transcriptional inhibition of gene expression or translation. Post-transcriptional genes silencing (PTGS) typically refers to the process of degradation or cleavage of messenger RNA (mRNA) molecules that decrease their activity by preventing translation. For example, a guide strand of an RNA silencing molecule pairs with a complementary sequence in a mRNA molecule and induces cleavage by various proteins, for example, Argonaute 2 (Ago2). Co-transcriptional gene silencing typically refers to inactivation of gene activity via transcription repression and typically occurs in the cell nucleus. Such gene activity repression is mediated by epigenetic-related factors, such as methyl-transferases, that methylate target DNAand histones. Thus, in co-transcriptional gene silencing, the association of a small RNA with a target RNA (small RNA-transcript interaction) destabilizes the target nascent transcript and recruits DNA- and hi stone-modifying enzymes that induce chromatin remodeling into a structure that repress gene activity and transcription. Also, in co-transcriptional gene silencing, chromatinAtty. Dkt. No. 102726-0300(TD-20-2024-W01)associated long non-coding RNA scaffolds may recruit chromatin-modifying complexes independently of small RNAs. These co-transcriptional silencing mechanisms form RNA surveillance systems that detect and silence inappropriate transcription events and provide a memory of these events via self-reinforcing epigenetic loops (see, e.g., Hoch, D. and Moazed. D., Nat. Rev. Genet., 2015, 16, 71-84).

[0034] As used herein, the terms “CD 163” and “Cluster of Differentiation 163” refers to nucleic acid sequences encoding a CD 163 protein, peptide, or polypeptide, or the amino acid sequence of the CD 163 protein, peptide, or polypeptide. Anon-limiting example of a CD163 transcript is Genbank Accession No. NM_213976, and any versions therefrom (i.e., NM_213976.1), which is incorporated by reference in its entirety. The term “CD163” may also include other CD 163 encoding sequences, such as CD 163 isoforms, mutant CD 163 genes, splice variants of CD 163 genes, and CD 163 gene polymorphisms, or the corresponding amino acid sequence therefrom. The term “CD163” may also refer to a nucleic acid sequence that encodes the polypeptide gene product of a CD 163 gene / transcript, e.g., a CD 163 protein, peptide, or polypeptide according to for example Genbank Accession No. NM_213976, and any versions therefrom (i.e., NM_213976.1).

[0035] The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof, in modified or unmodified form. Polynucleotides or nucleic acids can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides or nucleic acids: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, miRNA, siRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide or nucleic acid sequence can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide or nucleic acid can be further modified after polymerization,Atty. Dkt. No. 102726-0300(TD-20-2024-W01)such as by conjugation with a labeling component. Unless otherwise specified or required, any embodiment of this disclosure that is a nucleic acid or polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide or nucleic acid sequence is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide or nucleic acid is RNA. Thus, the term “polynucleotide sequence” or “nucleic acid sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.Polynucleotides or nucleic acids include, without limitation, single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- and doublestranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide or nucleic acid also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0036] As used herein, the phrase “silencing a target gene” refers to the absence or observable reduction in the level of protein and / or mRNA product from the target gene. Thus, silencing of a target gene can be by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% as compared to a target gene not targeted by the designed noncoding RNA molecule of the present disclosure. The consequences of silencing can be confirmed by examination of the outward properties of a eukaryotic cell or organism, or by biochemical techniques (as discussed herein). As used herein, gene silencing generally refers to a reduction in protein or mRNA product to result in an observable phenotype.

[0037] The term “siRNA” refers to small inhibitory RNA duplexes (generally between 18-30 base pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-baseAtty. Dkt. No. 102726-0300(TD-20-2024-W01)3 '-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is suggested to result from providing Dicer with a substrate (27mer) instead of a product (21mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC. It has been found that position, but not the composition, of the 3 '-overhang influences potency of a siRNA and asymmetric duplexes having a 3'-overhang on the antisense strand are generally more potent than those with the 3'-overhang on the sense strand (Rose, D., el al., Nucleic Acids Research, 2005, 33, 4140-4156).

[0038] The strands of a double-stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). Thus, as mentioned, the RNA silencing molecule of some embodiments of the present technology may also be a short hairpin RNA (shRNA).

[0039] The term “short hairpin RNA” and “shRNA,” as used herein, refers to a RNA molecule having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5'-CAAGAGA-3' and 5'-UUACAA-3' (International Patent Application Nos. WO2013126963 and WO2014107763). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.

[0040] As used herein, “gene editing” and “gene editing effectors” refer to the use of naturally occurring or artificially engineered nucleases, also referred to as “molecularAtty. Dkt. No. 102726-0300(TD-20-2024-W01)scissors.” The nucleases create specific double-stranded break (DSBs) at desired locations in the genome, which in some cases harnesses the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and / or nonhomologous end-joining (NHEJ). Gene editing effectors include Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the Clustered Regularly Interspaced Short Palindromic Repeats / CAS (CRISPR / Cas) system, and meganucleases re-engineered as homing endonucleases.

[0041] As used herein, the term “gene edit” or “gene edited” refers to an organism where human intervention, such as but without limitation by using a gene editing effector, has created a genetic difference in its genome when compared to a reference genome of the same organism. These differences can include but are not limited to nucleotide substitutions, excision of a start codon, or small deletions. In the present technology, a gene edit can be used to transform a wildtype non-coding nucleic acid sequence in a porcine host cell (e.g., a wildtype scaffold, such as a miRNA scaffold) into a GEiGS® solution (e.g., a gene-edited chromosomal sequence containing the GEiGS® modification) to generate a modified noncoding RNA molecule (e.g., siRNA) with a silencing specificity towards an endogenous CD 163 gene in porcine animals or porcine cells. In some embodiments, a gene edit does not introduce DNAfrom another species into an organism.

[0042] A “gene-edited animal” refers to an animal with one or more cells comprising a gene edit. In some embodiments, a gene edit can be a gene-edited chromosomal sequence. In some embodiments, a gene-edited chromosomal sequence can be a GEiGS® solution sequence as disclosed herein.

[0043] As used herein, a “pig” or “porcine animal” are used interchangeably and can be any member of the suid family (Family Suidae), including a domestic pig, a wild boar, a warthog, or any other porcine animal, including any domestic pig or domestic porcine animal (Sus scrofa domesticus). In some embodiments, a porcine animal is a sow. In some embodiments, a porcine animal is a boar. In some embodiments, a porcine animal is a barrow. In some embodiments a porcine animal is a piglet.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Gene Editing Methods of the Present Technology

[0044] The present disclosure provides a method for modulating CD 163 expression comprising contacting a pig cell with an effective amount of a DNA editing agent, wherein the DNA editing agent is configured to modify a gene or chromosomal sequence encoding or processed into a non-coding RNA molecule to generate a modified non-coding RNA molecule with a silencing specificity towards CD 163, thereby improving a trait in the pig. The methods disclosed herein are useful for generating gene-edited pigs that show an improvement in one or more traits or fitness without negatively affecting survival of the animals.

[0045] In some embodiments, the non-coding RNA molecule has no RNA silencing activity in the eukaryotic cell. In other embodiments, the non-coding RNA molecule has silencing specificity towards a first target RNA and the edited non-coding RNA molecule has silencing specificity towards a second target RNA, wherein the first target RNA and the second target RNA are distinct. Accordingly, the methods of the present technology are utilized to redirect a silencing activity and / or specificity of the non-coding RNA molecule (or to generate a silencing activity and / or specificity if the non-coding RNA molecule does not have an intrinsic capability to silence an RNA molecule) towards a second target RNA, which can be CD 163. Alternatively, in some embodiments, the modified non-coding RNA molecule has a silencing specificity towards one or more distinct target RNAs associated with CD 163. Moreover, the modified non-coding RNA molecules may be stably propagated to offspring without the need for further cross-breeding or gene editing events.

[0046] Alternatively or in addition, a cisgenic miRNAthat has been edited to express a GEiGS® solution is introduced to the cell. The sequence of the miRNA may be recapitulated in vitro with the sequence modified to target the mRNA of target gene. This sequence is then introduced into a target locus of the organism, such as but without limitation, the ROSA locus (e.g., the Rosa26 locus). In any embodiment herein, the GEiGS® solution sequence is targeted to the chromosomal location of an miRNA scaffold. For example, as described herein by the experimental examples (see, e.g., Examples 3, 4, and 5), the chromosomal target locus can be an miRNA scaffold. Exemplary miRNA scaffolds are presented in the second column ofAtty. Dkt. No. 102726-0300(TD-20-2024-W01)Table 2. For example, in some embodiments, the WT miRNA sequence (also referred to herein as the WT Solution Sequence) located within an miRNA scaffold, such as those shown in Table 2, is replaced by a GEiGS® solution sequence that encodes or is processed into an siRNA. Using the first embodiment listed in Table 2 as an example, the ssc-mir-155 chromosomal sequence as set forth in SEQ ID NO: 104 (the WT solution sequence shown in column 3 of Table 2) is edited to be replaced with the nucleotide sequence as set forth in SEQ ID NO: 1 thereby yielding a gene-edited chromosomal sequence that encodes a noncoding RNA (ncRNA) molecule capable of silencing an endogenous CD 163 gene, wherein the ncRNA molecule encodes or is processed into an siRNA having the nucleic acid sequence set forth in SEQ ID NO: 51. When expressed, this cisgenic miRNA can create knockdown or knockout of the target gene. In some embodiments, the GEiGS® solution sequence is one or more gene edited chromosomal sequences. In some embodiments, the one or more gene edited chromosomal sequences or one or more GEiGS® solution sequences comprise one or more nucleic acid sequences selected from SEQ ID NOs: 1-50. In some embodiments, the one or more gene edited chromosomal sequences or one or more GEiGS® solution sequences comprise one or more nucleic acid sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to one or more sequences selected from SEQ ID NOs: 1-50. In any of the embodiments described herein, the one or more gene edited chromosomal sequences or one or more GEiGS® solution sequences comprise one or more nucleic acid sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to one or more sequences selected from SEQ ID NOs: 1-50 and encode a non-coding RNA molecule (e.g, an siRNA) that silences an endogenous CD 163 gene.

[0047] Additionally or alternatively, in some embodiments, the eukaryotic cell may be a primary cell, a cell line, a somatic cell, a germ cell, a stem cell, an embryonic stem cell, an adult stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an induced pluripotent stem cell (iPSC), a gamete cell, a zygote cell, a blastocyst cell, an embryo, a fetus and / or a donor cell. In some embodiments, the eukaryotic cell may be isolated from its natural environment (e.g., animal body). Alternatively, in some embodiments, the eukaryotic cell may be a healthy cell, an immune cell including a T cell, B cell, macrophage, or a NK cell or a cell infected by a pathogen such as a bacterial, viral, or fungal pathogen. In variousAtty. Dkt. No. 102726-0300(TD-20-2024-W01)embodiments, the eukaryotic cell may be a PAM, a CPAM, an IPAM, or a MARC cell. In various embodiments, the eukaryotic cell may be any cell that may be transfected with the solution and tested for replication.

[0048] In some embodiments of the methods disclosed herein, the non-coding RNA molecule is typically subject to the RNA silencing processing mechanism or activity.However, also contemplated herein are a few changes in nucleotides (e.g., up to 24 nucleotides) which elicit a processing mechanism that results in RNA interference or translation inhibition. In various embodiments, larger edits, such as up to 50 nucleotides, up to 100 nucleotides, up to 150 nucleotides, or up to 200 nucleotides can be used to elicit the processing mechanism. Further modifications are discussed below. In certain embodiments, the non-coding RNA molecule is endogenous (naturally occurring, e.g., native) to the eukaryotic cell or exogenous to the cell ( / .< ., externally added and which is not naturally occurring in the cell). According to some embodiments, the non-coding RNA molecule comprises an intrinsic translational inhibition activity or an intrinsic RNAi activity. In other embodiments, the non-coding RNA molecule does not comprise an intrinsic translational inhibition activity or an intrinsic RNAi activity ( / .< ., the non-coding RNA molecule does not have an RNA silencing activity).

[0049] According to an embodiment of the present technology, the non-coding RNA molecule is specific to a target RNA (e.g., a natural target RNA) and does not cross inhibit or silence a second target RNA or target RNA of interest unless designed to do so (as discussed herein), exhibiting 100% or less global homology to the target gene, e.g, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, less than 82%, or less than 81% global homology to the target gene; as determined at the RNA or protein level. In some embodiments, methods for determining at the RNA or protein level can be either in silico or by RT-PCR, RT-qPCR, western blot, immunohistochemistry and / or flow cytometry or any other detection methods.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0050] Additionally or alternatively, in some embodiments of the methods disclosed herein, the non-coding RNA molecule is an RNA silencing or RNAi molecule. In some embodiments, the RNA silencing molecule is capable of mediating RNA repression during transcription (co-transcriptional gene silencing). According to a specific embodiment, co-transcri phonal gene silencing includes epigenetic silencing (e.g., a chromatic state that prevents gene expression). In some embodiments, the RNA silencing molecule is capable of modulating protein translation on other RNAs.

[0051] Additionally or alternatively, in some embodiments of the methods disclosed herein, the RNAi biogenesis / processing machinery generates the RNA silencing molecule.

[0052] Additionally or alternatively, in some embodiments of the methods disclosed herein, the non-coding RNA molecule is capable of inducing RNAi. According to one embodiment, the non-coding RNA molecule or the RNA silencing molecule is processed from a precursor. In certain embodiments, the non-coding RNA molecule or RNA silencing molecule is processed from a single stranded RNA(ssRNA) precursor, a duplex- structured single-stranded RNA precursor, a dsRNA precursor (e.g., comprising perfect and imperfect base pairing), a non-structured RNA precursor, a protein-coding RNA precursor, or a noncoding RNA precursor. According to one embodiment, the dsRNA is derived from two different complementary RNAs, or from a single RNA that folds on itself to form dsRNA.

[0053] Perfect and imperfect based paired RNA (i.e., double stranded RNA, dsRNA), siRNA and shRNA - The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). siRNAs derived from Dicer activity are typically about 1 to about 23 nucleotides in length and comprise about 19 base pair duplexes with two 3' nucleotides overhangs.

[0054] Accordingly, some embodiments of the present technology contemplate modifying a gene encoding a dsRNA to redirect a silencing specificity (including silencing activity) towards CD 163. According to one embodiment, dsRNA precursors longer than 21 bp areAtty. Dkt. No. 102726-0300(TD-20-2024-W01)used. Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects (see, e.g., Strat, A., etal., Nucleic Acids Research, 2006, 34, 3803-3810; Bhargava, A., et al., Brain Res. Protoc., 2004, 13, 115-125; Diallo, M., et al, Oligonucleotides, 2003, 13, 381-392;Paddison, P.J., et al., Proc. Natl. Acad. Sci. USA., 2002, 99, 1443-1448; Tran, N., et al., FEBS Lett., 2004, 573, 127-134).

[0055] The RNA silencing molecule of some embodiments of the present technology need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.

[0056] Various types of siRNAs are contemplated by the present technology, such as but without limitation, shRNAs. According to one embodiment, silencing RNA includes “piRNA,” which is a class of Piwi -interacting RNAs of about 26 and 31 nucleotides in length. piRNAs typically form RNA-protein complexes through interactions with Piwi proteins, i.e., antisense piRNAs are typically loaded into Piwi proteins (e.g., Piwi, Ago3 and Aubergine (Aub)).

[0057] According to some embodiments, the RNA silencing molecule is a miRNA. A number of studies have looked at the base-pairing requirement between miRNA and its mRNA target for achieving efficient inhibition of translation (reviewed by Bartel, D.P, Cell, 2004, 116, 281-297). Computational studies analyzing miRNA binding on whole genomes have suggested a specific role for bases 2-8 at the 5' of the miRNA (also referred to as “seed sequence”) in target binding, but the role of the first nucleotide, found usually to be “A” was also recognized (Lewis, B.P, etal., Cell, 2005, 120, 15-20). Similarly, nucleotides 1-7 or 2-8 were used to identify and validate targets (Krek, A., et al., Nat. Genet., 2005, 37, 495-500). The target sites in the mRNA may be in the 5' UTR, the 3' UTR, or in the coding region. Interestingly, multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites. The presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RNA-induced silencing complexes (RISCs) provides the most efficient translational inhibition.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0058] miRNAs may direct the RISCs to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression. The miRNAmay specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut is typically between the nucleotides pairing to residues 10 and 11 of the miRNA. Alternatively, the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.

[0059] It should be noted that there may be variability in the 5' and 3' ends of any pair of miRNA and miRNA* ( / .< ., the minor arm of the miRNA strand). This variability may be due to variability in the enzymatic processing of Drosha and Dicer with respect to the site of cleavage. Variability at the 5' and 3' ends of miRNA and miRNA* may also be due to mismatches in the stem structures of the pri-miRNA and pre-miRNA. The mismatches of the stem strands may lead to a population of different hairpin structures. Variability in the stem structures may also lead to variability in the products of cleavage by Drosha and Dicer.

[0060] It will be appreciated that the pre-miRNA sequence may comprise from 45-90, 60-80, or 60-70 nucleotides while the pri-miRNA sequence may comprise from 45-30,000, SO-25, 000, 100-20,000, 1,000-1,500, or 80-100 nucleotides.

[0061] Antisense RNA - Antisense RNA is a single stranded RNA designed to prevent or inhibit expression of a gene by specifically hybridizing to its mRNA. Downregulation of a target RNA can be effected using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the target RNA.

[0062] As mentioned herein, the non-coding RNA molecule may not comprise a canonical (intrinsic) RNAi activity (e.g., is not a canonical RNA silencing molecule, or its target has not been identified).

[0063] According to some embodiments, the non-coding RNA molecule is a transfer RNA (tRNA). The term “tRNA” refers to an RNA molecule that serves as the physical link between nucleotide sequence of nucleic acids and the amino acid sequence of proteins,Atty. Dkt. No. 102726-0300(TD-20-2024-W01)formerly referred to as soluble RNA or sRNA. tRNA is typically about 76 to 90 nucleotides in length.

[0064] According to some embodiments, the non-coding RNA molecule is a ribosomal RNA (rRNA). The term “rRNA” refers to the RNA component of the ribosome — either the small ribosomal subunit or the large ribosomal subunit.

[0065] According to some embodiments, the non-coding RNA molecule is a small nuclear RNA (snRNA or U-RNA). The terms “sRNA” or “U-RNA” refer to the small RNA molecules found within the splicing speckles and Cajal bodies of the cell nucleus in eukaryotic cells. snRNA is typically about 150 nucleotides in length.

[0066] According to some embodiments, the non-coding RNA molecule is a small nucleolar RNA (snoRNA). The term “snoRNA” refers to the class of small RNA molecules that primarily guide chemical modifications of other RNAs, e.g., rRNAs, tRNAs and snRNAs. snoRNA is typically classified into one of two classes: the C / D box snoRNAs are typically about 70-120 nucleotides in length and are associated with methylation, and the H / ACAbox snoRNAs are typically about 100-200 nucleotides in length and are associated with p seudouri dy 1 ati on .

[0067] Similar to snoRNAs are the scaRNAs (i.e. Small Cajal body RNA genes) which perform a similar role in RNA maturation to snoRNAs, but their targets are spliceosomal snRNAs and they perform site-specific modifications of spliceosomal snRNA precursors (in the Cajal bodies of the nucleus).

[0068] According to some embodiments, the non-coding RNA molecule is an extracellular RNA (exRNA). The term “exRNA” refers to RNA species present outside of the cells from which they were transcribed (e.g., exosomal RNA).

[0069] According to some embodiments, the non-coding RNA molecule is a long noncoding RNA (IncRNA). The term “IncRNA” or “long ncRNA” refers to non-protein coding transcripts typically longer than 200 nucleotides.1Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0070] Additionally or alternatively, in any of the preceding embodiments, non-limiting examples of non-coding RNA molecules include, but are not limited to, microRNA (miRNA), piwi-interacting RNA (piRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), small nuclear RNA (snRNA or URNA), small nucleolar RNA (snoRNA), Small Cajal body RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), extracellular RNA (exRNA), repeat-derived RNA, transposable element RNA, and long non-coding RNA (IncRNA).

[0071] According to some embodiments, non-limiting examples of RNAi molecules include, but are not limited to, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), and trans-acting siRNA (tasiRNA).

[0072] As mentioned above, the methods of some embodiments of the present technology are utilized to redirect a silencing activity and / or specificity of the noncoding RNA molecule (or to generate a silencing activity and / or specificity if the noncoding RNA molecule does not have an intrinsic capability to silence an RNA molecule) towards a second target RNA or towards a target RNA of interest.

[0073] In some embodiments, the second target RNA or target RNA of interest is endogenous to the eukaryotic cell. Exemplary endogenous second target RNAs or target RNAs of interest include CD 163.

[0074] The specific binding of an endogenous non-coding RNA molecule with a target RNA can be determined by computational algorithms (such as BLAST) and verified by methods including e.g., northern blot, in situ hybridization, QU ANTIGENE® Plex Assay (ThermoFisher Scientific, Waithem, MA), etc.

[0075] Additionally or alternatively, in some embodiments, if the non-coding RNA molecule is or is processed into a siRNA, the complementarity is in the range of 90% -100% (e.g., 100%) to its target sequence. Additionally or alternatively, in some embodiments, if the non-coding RNA molecule is a miRNA, the seed sequence complementarity (i.e. nucleotides 2-8 from the 5') is in the range of 85%-100% (e.g., 100%)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)to its target sequence. In general, the complementarity is measured in terms of the mature siRNA form, instead of the precursor form.

[0076] According to one embodiment, the non-coding RNA molecule (i.e. prior to modification) is typically selected as one having about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or up to about 99% complementarity towards the sequence of the CD 163 gene.

[0077] In order to generate silencing activity and / or specificity of a non-coding RNA molecule or redirect a silencing activity and / or specificity of a non-coding RNA molecule (e.g., RNA silencing molecule) toward CD 163, the gene encoding a non-coding RNA molecule (e.g., RNA silencing molecule) is modified using a DNA editing agent. These include meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs), and the CRISPR / Cas9 system (and variants thereof).

[0078] CRISPR-Cas system and all its variants (also referred to herein as “CRISPR ”) -Many bacteria and archaea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) nucleotide sequences that produce RNA components and CRISPR associated (Cas) genes that encode protein components. The CRISPR RNAs (crRNAs) contain short stretches of homology to the DNA of specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen. Studies of the type II CRISPR / Cas system of Streptococcus pyogenes have shown that three components form a RNA / protein complex and that together are sufficient for sequence-specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans activating crRNA (tracrRNA) (Jinek, M., et al., Science, 2012, 337:816-821).

[0079] It was further demonstrated that a synthetic chimeric guide RNA (gRNA) composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary to the crRNA in vitro. It was also demonstrated that transient expression ofAtty. Dkt. No. 102726-0300(TD-20-2024-W01)Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double-stranded breaks (DSBs) in a variety of different species.

[0080] The CRISPR / Cas system for genome editing contains two distinct components: a gRNA and a CRISPR endonuclease. A variety of CRISPR endonucleases are available for use in conjunction with the gRNA. In some embodiments, the CRISPR enzyme can be a Type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme can be any Cas9 protein, for instance any naturally occurring bacterial Cas9 as well as any chimeras, mutants, homologs or orthologs.

[0081] The gRNA (also referred to herein as single guide RNA (sgRNA)) typically encodes a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA / Cas9 complex can be recruited to the target sequence by the base pairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA / Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break (DSB). The double-stranded breaks (DSBs) produced by CRISPR / Cas can undergo homology-directed repair or NHEJ and are susceptible to specific sequence modification during DNA repair.

[0082] There are a number of publicly available tools available to help choose and / or design target sequences as well as lists of bioinformatically determined unique gRNAs for different genes in different species, such as but not limited to, E-CRISP (Boutros Lab, German Cancer Research Center), the CRISPR RGEN Tools: CasOFFinder (BAE Lab, Seoul National University College of Medicine, South Korea), the CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes and the CRISPR Optimal Target Finder (Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA).

[0083] In order to use the CRISPR system, both gRNA and a Cas endonuclease (e.g., Cas9) should be expressed or present (e.g., as a ribonucleoprotein complex) in a target cell. TheAtty. Dkt. No. 102726-0300(TD-20-2024-W01)insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids. CRISPR plasmids are commercially available such as the px330 plasmid from ADDGENE® (75 Sidney St, Suite 550A, Cambridge, MA 02139). Use of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology and a Cas endonuclease for modifying mammalian genomes are also at least disclosed by Bauer , D.E., etal., (Vis. Exp., 2015, 95, e52118. doi: 10.3791 / 52118), which is specifically incorporated herein by reference in its entirety. Cas endonucleases that can be used to effect DNA editing with gRNA include, but are not limited to, Cas9, Cpfl (Zetsche, B, etal., Cell, 2015, 163, 759-771), C2cl, C2c2, and C2c3 (Shmakov, S., etal., Mol. Cell., 2015, 60, 385-397).

[0084] According to a some embodiments, the DNA editing agent comprises a DNA targeting module e.g., gRNA). The DNA editing agent may or may not further comprise an endonuclease. In some embodiments of the method, the DNA editing agent comprises a nuclease e.g., an endonuclease) and a DNA targeting module (e.g., gRNA). In certain embodiments, the DNA editing agent is CRISPR / Cas, e.g., gRNA and Cas9.

[0085] According to a some embodiments, the DNA editing agent is TALEN. According to some embodiments, the DNA editing agent is ZFN. According to some embodiments, the DNA editing agent is a meganuclease.

[0086] Regardless of the DNA editing agent used, the methods of the present technology are employed such that the gene encoding the non-coding RNA molecule (e.g., RNA silencing molecule) is modified by at least one of a deletion, an insertion, or a point mutation.

[0087] According to some embodiments, the modification is in a structured region of the non-coding RNA molecule or the RNA silencing molecule. In some embodiments, the modification is in a stem region of the non-coding RNA molecule or the RNA silencing molecule. In some embodiments, the modification is in a loop region of the noncoding RNA molecule or the RNA silencing molecule. In other embodiments, the modification is in a stem region and a loop region of the non-coding RNA molecule or the RNA silencing molecule.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0088] According to some embodiments, the modification is in a non- structured region of the non-coding RNA molecule or the RNA silencing molecule. In some embodiments, the modification is in a stem region and a loop region and in non- structured region of the noncoding RNA molecule or the RNA silencing molecule.

[0089] Additionally or alternatively, in some embodiments, the modification depends on the structure of the RNA silencing molecule. Accordingly, when the RNA silencing molecule contains a non-essential structure (z.e., a secondary structure of the RNA silencing molecule which does not play a role in its proper biogenesis and / or function) or is purely dsRNA (z.e., the RNA silencing molecule having a perfect or almost perfect dsRNA), a few modifications (e.g., 20-30 nucleotides, e.g., 1-10 nucleotides, e.g., 5 nucleotides) are introduced in order to redirect the silence specificity of the RNA silencing molecule.

[0090] According to some embodiments, when the RNA silencing molecule has an essential structure (z.e., the proper biogenesis and / or activity of the RNA silencing molecule is dependent on its secondary structure), larger modifications (e.g., 10-200 nucleotides, e.g., 50-150 nucleotides, e.g., more than 30 nucleotides and not exceeding 200 nucleotides, 30-200 nucleotides, 35-200 nucleotides, 35-150 nucleotides, 35-100 nucleotides) are introduced in order to redirect the silence specificity of the RNA silencing molecule.

[0091] According to some embodiments, the modification can be such that the recognition / cut site / PAM motif of the RNA silencing molecule is modified to abolish the original PAM recognition site. According to some embodiments, the modification is in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids in a PAM motif.

[0092] Additionally or alternatively, in some embodiments, the modification comprises an insertion. According to some embodiments, the insertion can comprise about 1-250 nucleotides (as compared to the native non-coding RNA molecule, e.g., RNA silencing molecule).

[0093] Additionally or alternatively, in some embodiments, the modification comprises a deletion. According to some embodiments, the deletion comprises about 1-250 nucleotides (as compared to the native non-coding RNA molecule, e.g., RNA silencing molecule).Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0094] Additionally or alternatively, in some embodiments, the modification comprises a combination of any of a deletion, an insertion, and / or one or more single nucleotide substitutions.

[0095] Additionally or alternatively, in some embodiments, the modification comprises nucleotide replacement (e.g., nucleotide swapping).

[0096] Additionally or alternatively, in certain embodiments, the gene encoding the noncoding RNA molecule (e.g., RNA silencing molecule) is modified by swapping a sequence of an endogenous RNA silencing molecule (e.g., miRNA) with an RNA silencing sequence of choice (e.g., siRNA).

[0097] Additionally or alternatively, in certain embodiments, the guide strand and / or the passenger strand of the non-coding RNA molecule (e.g., RNA silencing molecule) is modified to preserve originality of structure and keep the same base pairing profile. As used herein, the term “originality of structure” refers to the secondary RNA structure (i.e., base pairing profile). Keeping the originality of structure can be important for correct and efficient biogenesis / processing of the non-coding RNA (e.g., RNA silencing molecule such as siRNA or miRNA) that is structure- and not purely sequence-dependent.

[0098] Additionally or alternatively, in some embodiments, the non-coding RNA (e.g., RNA silencing molecule) is modified in the guide strand (silencing strand) so as to comprise about 50% to aboutl00% complementarity to the target RNA while the passenger strand is modified to preserve the original (unmodified) non-coding RNA structure. According to some embodiments, the non-coding RNA (e.g., RNA silencing molecule) is modified such that the seed sequence (e.g., for miRNA nucleotides 2-8 from the 5' terminal) is complementary to the target sequence.

[0099] According to a specific embodiment, the RNA silencing molecule is designed such that a sequence of the RNAi molecule is modified to preserve originality of structure and to be recognized by cellular RNAi processing and executing factors.

[0100] Delivery ofDNA editing agents . Additionally or alternatively, in some embodiments of the methods disclosed herein, the DNA editing agent is introduced into eukaryotic cellsAtty. Dkt. No. 102726-0300(TD-20-2024-W01)using DNA delivery methods (e.g., by expression vectors) or using DNA-free methods. According to some embodiments, the gRNA (or any other DNA recognition module used, dependent on the DNA editing system that is used) is provided as RNA to the cell.

[0101] Thus, it will be appreciated that the present techniques relate to introducing the DNA editing agent using DNA-free methods such as RNA transfection (e.g., mRNA+gRNA transfection), or Ribonucleoprotein (RNP) transfection (e.g., protein-RNA complex transfection, e.g., Cas9 / gRNARNP complex transfection, or any combination of DNA / RNA / Proteins). For example, Cas9 can be introduced as a DNA expression plasmid, in vitro transcript (i.e., RNA), or as a recombinant protein bound to the RNA portion in a ribonucleoprotein particle (RNP). gRNA, for example, can be delivered either as a DNA plasmid or as an in vitro transcript (i.e., RNA).

[0102] Any method known in the art for RNA or RNP transfection can be used in accordance with the present teachings, such as, but not limited to microinjection (see, e.g., Cho, W.C., etaL, Genetics, 2013, 195, 1177-1180), electroporation (see e.g., Kim, S., etaL, Genome Res., 2014, 24, 1012-1019), or lipid-mediated transfection e.g., using liposomes (see e.g., Zuris, J. A., et al., Nat. Biotechnol., 2014, 33, 73-80). Additional methods of RNA transfection are described in U.S. Patent Application No. 20160289675.

[0103] One advantage of RNA transfection methods of the present technology is that RNA transfection can be essentially transient and vector-free. An RNA transgene is delivered to a cell and expressed therein, as a minimal expressing cassette without the need for any additional sequences (e.g., viral sequences).

[0104] According to some embodiments, for expression of exogenous DNA editing agents of the present technology in mammalian cells, a polynucleotide sequence encoding the DNA editing agent is ligated into a nucleic acid construct suitable for mammalian cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner. Expression vectors are known in the art, many of which are available commercially. It is within the expertise of the skilled person to select an appropriate expression vector.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0105] Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBKRSV and pBK-CMV which are available from StrataGene®, pTRES which is available from CLONETECH Laboratories, Inc, and their derivatives.

[0106] Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0107] Viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by some embodiments of the present technology will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein. For example, bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) (Liang, C.Y, el al., Arch Virol., 2004, 149, 51-60).

[0108] Recombinant viral vectors can be useful for in vivo expression of DNA editing agents since they offer advantages such as lateral infection and targeting specificity. LateralAtty. Dkt. No. 102726-0300(TD-20-2024-W01)infection can be inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area can become rapidly infected, most of which was not initially infected by the original viral particles. This contrasts with vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0109] Alternatively, when a nuclease is not utilized ( / .< ., not administered from an exogenous source to the cell), the DNA recognition unit (e.g., gRNA) may be cloned and expressed using a single expression vector.

[0110] According to some embodiments, the DNA editing agent can comprise a nucleic acid agent encoding at least one DNA recognition unit (e.g., gRNA) operatively linked to a cisacting regulatory element active in eukaryotic cells (e.g, promoter). In other embodiments of the methods disclosed herein, the DNA editing agent can comprise at least one gRNA that is not linked operatively linked to a promoter.

[0111] According to some embodiments, the nuclease (e.g, endonuclease) and the DNA recognition unit (e.g., gRNA) can be encoded from the same expression vector. Such a vector may comprise a single cis-acting regulatory element active in eukaryotic cells (e.g., promoter) for expression of both the nuclease and the DNA recognition unit. Alternatively, the nuclease and the DNA recognition unit may each be operably linked to a cis-acting regulatory element active in eukaryotic cells (e.g., promoter).

[0112] According to some embodiments, the nuclease (e.g., endonuclease) and the DNA recognition unit (e.g., gRNA) can be encoded from different expression vectors whereby each is operably linked to a cis-acting regulatory element active in eukaryotic cells (e.g., promoter).

[0113] In various embodiments, an optional polynucleotide template for HDR repair can be introduced into the cell with the endonuclease and DNA recognition unit. This repairAtty. Dkt. No. 102726-0300(TD-20-2024-W01)template can be used by the cell as a template when fixing the double stranded break and thus can be used to direct changes in the DNA sequence.

[0114] Various methods can be used to introduce the expression vector or donor oligonucleotides of some embodiments of the present technology into eukaryotic cells (e.g., stem cells). Such methods are generally described in Sambrook, etal., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel, et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega, etal., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa, E., et al., (Biotechniques, 1986, 4, 504-512), and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0115] Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

[0116] In vivo nucleic acid transfer techniques include transfection with viral or nonviral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi (Tonkinson, J.L. and Stein, C.A., Cancer Investigation, 1996, 14, 54-65). For gene therapy, the preferred constructs are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct can include at least one transcriptional promoter / enhancer or locus defining element(s), or other elements that controls gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which itAtty. Dkt. No. 102726-0300(TD-20-2024-W01)is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the present technology. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.

[0117] Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of some embodiments of the present technology can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide.

[0118] According to a specific embodiment, a bombardment method is used to introduce foreign genes into eukaryotic cells. According to one embodiment, the method is transient. Bombardment of eukaryotic cells (e.g., mammalian cells) is also taught by Uchida, M., et aL, Biochim. Biophys. Acta.., 2009, 1790, 754-764.

[0119] Regardless of the transformation / infection method employed, the methods as disclosed herein, further include selecting transformed cells comprising a genome editing event. According to some embodiments, selection can be carried out such that only cells comprising a successful accurate modification (e.g, swapping, insertion, deletion, nucleotide substitution) in the specific locus are selected. Accordingly, in some embodiments, cells comprising any event that includes a modification (e.g, an insertion, deletion, nucleotide substitution) in an unintended locus are not selected.

[0120] According to some embodiments, selection of modified cells is performed at the phenotypic level, by detection of a molecular event, by detection of a fluorescent reporter, or by growth in the presence of selection (e.g., antibiotic or other selection marker such as resistance to a drug).

[0121] According to some embodiments, selection of modified cells is performed by analyzing the biogenesis and occurrence of the newly edited non-coding RNA moleculeAtty. Dkt. No. 102726-0300(TD-20-2024-W01)e.g., the presence of new miRNA version, the presence of novel edited siRNAs, piRNAs, tasiRNAs, etc).

[0122] According to some embodiments, selection of modified cells is performed by analyzing the silencing activity and / or specificity of the non-coding RNA molecule (e.g., RNA silencing molecule) towards a CD 163 by validating phenotype such as viral resistance for CD 163.

[0123] According to some embodiments, the silencing specificity of the non-coding RNA molecule is determined genotypically, e.g., by expression of a gene or lack of expression.

[0124] According to some embodiments, selection of modified cells is performed by analyzing the silencing activity and / or specificity of the non-coding RNA molecule (e.g., RNA silencing molecule) towards CD 163. This can be effected using any method known in the art, e.g., by northern blotting, nuclease protection assays, in situ hybridization, quantitative real time polymerase chain reaction (qRT-PCR), or immunoblotting.

[0125] Methods for detecting sequence alteration are well known in the art and include, but are not limited to, DNA and RNA sequencing (e.g., next generation sequencing), electrophoresis, an enzyme-based mismatch detection assay and a hybridization assay such as PCR, RT-PCR, RNase protection, in situ hybridization, primer extension, Southern blot, northern blot and dot blot analysis. Various methods used for detection of single nucleotide polymorphisms (SNPs) can also be used, such as PCR based T7 endonuclease, heteroduplex mobility assay, Sanger sequencing, or PCR followed by restriction digest to detect appearance or disappearance of unique restriction site / s.

[0126] Another method of validating the presence of a DNA editing event, e.g., indels comprises a mismatch cleavage assay that makes use of a structure selective enzyme (e.g., endonuclease) that recognizes and cleaves mismatched DNA.

[0127] The specific binding of designed non-coding RNA molecule with CD 163 can be determined by any method known in the art, such as by computational algorithms (e.g., BLAST) and verified by methods such as, but without limitation, northern blot, in situ hybridization, or QU ANTIGENE™ Plex Assay.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0128] It will be appreciated that positive eukaryotic cells can be homozygous or heterozygous for the DNA editing event. In the case of a heterozygous cell, the cell may comprise a copy of a modified gene and a copy of a non-modified gene of the non-coding RNA molecule (e.g., RNA silencing molecule). The skilled artisan will select the cells for further culturing / regeneration according to the intended use.

[0129] According to some embodiments, when a transient method is desired, eukaryotic cells exhibiting the presence of a DNA editing event as desired are further analyzed and selected for the presence of the DNA editing agent, namely, loss of DNA sequences encoding for the DNA editing agent. This can be done, for example, by analyzing the loss of expression of the DNA editing agent (e.g., at the mRNA, protein) e.g., by fluorescent detection of GFP or qRT-PCR, HPLC.

[0130] According to some embodiments, when a transient method is desired, the eukaryotic cells are analyzed for the presence of the nucleic acid construct as described herein or portions thereof, e.g., nucleic acid sequence encoding the DNA editing agent. This is affirmed by fluorescent microscopy, qRT-PCR, fluorescence-activated cell sorting (FACS), and / or by any other method such as Southern blot, PCR, sequencing, HPLC. Positive eukaryotic cell clones may be stored (e.g., cryopreserved).

[0131] Alternatively, eukaryotic cells may be further cultured and maintained, for example, in an undifferentiated state for extended periods of time or may be induced to differentiate into other cell types, tissues, organs, or organisms as required.

[0132] The DNA editing agents and optionally the donor oligonucleotides of some embodiments of the present technology can be administered to a single cell, to a group of cells (e.g., primary cells or cell lines as discussed above), or to a porcine animal ( / .< ., a pig).Uses of the Gene Editing Agents of the Present Technology

[0133] In one aspect, the present disclosure provides a method for improving fitness in pigs, the method comprising contacting a pig cell with an effective amount of a DNA editing agent, wherein the DNA editing agent is configured to modify a gene encoding or processedAtty. Dkt. No. 102726-0300(TD-20-2024-W01)into a non-coding RNA molecule to generate a modified non-coding RNA molecule with a silencing specificity towards CD 163.

[0134] Additionally or alternatively, in some embodiments, the modified non-coding RNA molecule silences the target RNA encoded by CD 163 in all cell types or within specific cell types of pigs. Examples of specific cell types include, but are not limited to, epidermal cells, lung tissue cells, intestinal cells, liver cells, pancreatic cells, blood cells, renal cells, neuronal cells, muscle cells, or immune cells such as macrophages, including pulmonary alveolar macrophages.

[0135] In another aspect, the DNA editing agent is designed to confer a silencing specificity of a non-coding RNA molecule towards CD 163 in pigs.

[0136] According to one aspect of the present technology, provided is a method of improving one or more traits in pigs in need thereof, the method comprising modifying a gene encoding or processed into a non-coding RNA molecule or encoding or processed into an RNA silencing molecule according to the method of some embodiments of the present technology, wherein the target RNA of interest is encoded by CD 163.

[0137] According to one aspect of the present technology, provided is a DNA editing agent conferring a silencing specificity to a non-coding RNA molecule having no RNA silencing activity towards a target RNA of interest, wherein the target RNA of interest is associated with CD 163, for use in improving the one or more traits in pigs in need thereof.

[0138] According to one aspect of the present technology, provided is a DNA editing agent redirecting a silencing specificity of a gene encoding or processed into a RNA silencing molecule from a first target RNA towards a second target RNA, the first target RNA and the second target RNA being distinct, wherein the second target RNA is CD163 in pigs.

[0139] According to one embodiment, the target RNA of interest comprises a product of the CD 163 gene of the pig cell conferring resistance to PRRSv.

[0140] Exemplary nucleic acid sequences for CD163 in pigs are listed in Table 1.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Table 1Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0141] As disclosed herein, endogenous non-coding RNA molecules may be modified to target a CD 163 RNA, using gRNA sequences (i.e., a DNA editing agent). Alternatively, the silencing specificity of an endogenous non-coding RNA molecule may be redirected towards CD 163. Exemplary changes are listed as they appear in the genome and the mature sequences are included as DNA for ease of comparison for the reader. It is noted that several precursor sequences result in the same predicted mature sRNA. A person of ordinary skill inAtty. Dkt. No. 102726-0300(TD-20-2024-W01)the art would know how to convert either of these sequences into an RNA sequence to determine the pre-miRNA or mature miRNA sequence.

[0142] Exemplary changes targeting CD 163 are listed in Table 2. These changes are listed as DNA for reasons described above.Table 2Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)* With reference to Table 2, the WT Solution Sequence is also referred to as the WT miRNA Sequence.#WT miRNA Sequences and GEiGS Solution Sequences are expressed transcripts which form hairpins, which are then cleaved into double-stranded duplexes of guide sRNAs and passenger sRNAs. In the overall GEiGS Solution Sequence, therefore, there are sequences corresponding to the guide and the passenger sequence; the passenger does not participate in silencing activity against the intended target (z. e. , CD 163), but differs from the WT sequence because it is required to form the hairpin with the guide sequence. The predicted mature guide sRNA is represented within the GEiGS Solution Sequence column as lowercase." For the purposes of the WT mature sRNA (guide) and Predicted mature sRNA(guide) sequences presented in columns 4 and 6 of Table 2, respectively, the thymine (T) residues would be understood by a person of ordinary skill in the art to be uracil (U) residues in an RNA sequence. They are presented in Table 2 as DNA for ease of comparison for the reader.Table 3Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Table 4Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0143] In various embodiments of the methods disclosed herein, the modified non-coding RNA molecules are complementary to a portion of a double-stranded DNA molecule comprising a nucleic acid sequence as set forth in any one of SEQ ID NOs: 1-50 that encodes a mRNA sequence.

[0144] The modified non-coding RNA molecules are complementary to a portion of a protein coding region (one or more exons) of a double-stranded DNA molecule. Additionally or alternatively, in some embodiments, the modified non-coding RNA molecules are complementary to a portion of a noncoding region of a double-stranded DNA molecule comprising a nucleic acid sequence as set forth in any one of SEQ ID NOs: 1-50. In some embodiments, the noncoding region refers to an exon-intron junction sequence, or the 5'Atty. Dkt. No. 102726-0300(TD-20-2024-W01)and / or 3' untranslated regions that flank the coding region and are not translated into amino acids. For example, the modified non-coding RNA molecule can be complementary to the region surrounding the translation start site of a mRNA sequence encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOs: 1-50.

[0145] The modified non-coding RNA molecules are generated in situ such that they hybridize with or bind to a CD 163 RNA to inhibit expression of CD 163.

[0146] Additionally or alternatively, in some embodiments, expression of the modified noncoding RNA molecule is regulated by operably linking the modified non-coding RNA molecule to a gene regulatory sequence (e.g., promoter or enhancers). The gene regulatory sequence may be a heterologous or an endogenous gene regulatory sequence (e.g., promoter or enhancer). Additionally or alternatively, in some embodiments, the gene regulatory sequence (e.g., promoter or enhancer) is constitutive or inducible. Additionally or alternatively, in some embodiments, the gene regulatory sequence drives ubiquitous expression of the modified non-coding RNA molecule, or limits expression of the modified non-coding RNA molecule in cell type-specific or a tissue specific manner. In certain embodiments, the gene regulatory sequence drives tissue-specific expression of the modified non-coding RNA molecule in one or more of lungs, pulmonary alveolar macrophage cells, or in muscle cells or muscle tissues.

[0147] Examples of tissue-specific promoters include promoters such as muscle-specific promoters such as muscle creatine kinase (MCK) promoter; lung-specific promoters such as surfactant protein B, surfactant protein A, surfactant protein C, and Clara cell secretory protein (CCSP) gene promoters. Alternatively, or in addition, the GEiGS® solution may be expressed using the endogenous promoter of its miRNA scaffold.

[0148] Designing GEiGS® sequences requires: a) a target sequence to be silenced by Gene Editing-induced Gene Silencing (GEiGS®) (“target”); b) choosing whether the GEiGS® (i.e., the modified non-coding RNA) would be expressed ubiquitously (e.g., constitutively) or conditionally (e.g., expression specific to a certain tissue, developmental stage, stress, heat / cold shock efc.).Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0149] Selection of the non-coding RNA depends upon the endogenous sequence to be edited and can be achieved by querying publicly available miRNA datasets (e.g., small RNA sequencing, genomic sequences, microarrays, efc.) so as to filter only relevant miRNAs that match the input criteria: miRNAs that are expressed according to the requirement(s) described above.

[0150] Using publicly available tools, a list of potent target-specific siRNA sequences is generated. The miRNAs are aligned against the potent siRNA sequences and the most homologous miRNAs are elected. Filtered miRNAs may have a similar sequence in the same orientation as the potent siRNAs.

[0151] Precursor miRNA sequences are edited to ensure that the mature miRNA produced by that sequence perfectly matches the sequence of CD 163. This editing occurs in one mature miRNA strand with the highest target homology (e.g., either the original miRNA guide or passenger strand). Such 100% complementary to the target can potentially turn the miRNA sequence into an siRNA.

[0152] Minimal genome editing is achieved by filtering miRNA sequences with naturally occurring high homology (reverse complement) to CD 163. The modified miRNA's guide strand (silencing strand) sequence is designed to be 100% complementary to the target. Editing is tuned so that only one allele of the miRNA sequence is edited; that is, the animal is heterozygous for the gene edit.

[0153] sgRNAs can be designed to specifically target the original unmodified miRNA gene (specific to the genomic miRNA loci), and not the modified version ( / .< ., the oligo / fragment sequences).

[0154] Validation. Examining the targeting of the non-coding RNA towards other targets (e.g., “off target effect”), using in silico methods, when the endogenous non-coding RNA (e.g., miRNA) comprises naturally occurring high homology with the target (e.g., 6090), so as to obtain specific silencing of the target of interest.

[0155] Minimally modifying the endogenous non-coding RNA (e.g., miRNA) to boost its potency to silence the target of interest.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0156] Validating GEiGS® outcome of the primary minimally edited miRNA genes to generate candidate refined minimally edited miRNAs. An experimentally effective primary GEiGS® outcome (the primary minimally edited miRNA genes) is considered a miRNA(s) with a guide or passenger strand that is modified to match CD 163.

[0157] Keeping the seed sequence in a way that there are at least 5 matches out of the seven seed nucleotides (nucleotides 2-8 from the 5' terminus).

[0158] Testing the various candidate “refined minimally edited miRNA genes” for CD 163 silencing efficiency. Choosing the GEiGS® solution that provides the highest silencing with the least miRNA sequence modifications.

[0159] Testing potential “off target effects” of refined minimally edited miRNA candidates. A significant prediction for “off target effects” affects the final evaluation of the refined minimally edited miRNA genes.

[0160] Once non-coding RNAs are identified, their sequences are engineered such that they silence expression of CD 163 transcripts. Sequence engineering can be performed manually, or computationally using, for example, GEiGS® BioCompute. Silencing solutions are tested in cells which express the silencing target sequence naturally or artificially. Silencing solutions are expressed either transiently or stably from a plasmid under control of a constitutive or regulatable promoter, or are integrated into the genome either in a native context, in a safe harbor locus or other prescribed location, or randomly using, for example, lentivirus.

[0161] The efficacy of a solution in silencing an RNA molecule is measured directly using RT-qPCR, for example, with primers directed against CD 163. In this case, an increase in the Cq (quantification cycle) value for the silencing target is indicative of gene silencing.Alternatively, where reporter systems are used, the reporter gene can be monitored directly by RT-qPCR or indirectly by fluorescence, luminescence, or another method depending on the reporter gene.

[0162] Once an efficacious silencing solution has been identified, the solution sequence is integrated into the porcine genome at the native non-coding RNA locus, or in a safe harborAtty. Dkt. No. 102726-0300(TD-20-2024-W01)site or other prescribed location. One method for such integration includes, but is not limited to, use of Cas9 and HDR-mediated repair. Alternatively, a silencing solution sequence is integrated randomly into the genome using lentivirus or other delivery system. Once edited animals have been generated, resistance to PRRSv in edited animals is evaluated in live virus animal challenges or cell-based assays using, but not limited to, monocyte-derived macrophages or pulmonary alveolar macrophages.EXAMPLES

[0163] It is to be understood that the present technology is not necessarily limited in its application to the details set forth in the description or exemplified by the Examples. The present technology is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0164] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims.

[0165] The following methods are used throughout the examples.Cell culture

[0166] hTERT-immortalized porcine pulmonary alveolar macrophages (CPAMs; abm T0741) were grown in Roswell Park Memorial Institute (RPMI) 1640 medium, ATCC modification (Gibco A1049101) supplemented with 10% (v / v) fetal bovine serum (FBS), 1% (v / v) minimum essential medium non-essential amino acids (MEM-NEAA), and 1% (v / v) penicillin-streptomycin (PS). Cells were maintained at 37°C, 5% CO2.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)siRNA transfection

[0167] CD 163 GEiGS® solutions were ordered as 21-23 nt custom siRNAs with 3’ UU dinucleotide overhangs on the antisense strand (Horizon Discovery). All siRNAs were resuspended in siRNA Buffer (300 mM KC1, 30 mM HEPES-pH 7.5, 1.0 mM MgCh; Horizon Discovery B-002000-UB). Cells were plated in triplicate at 1.50 x 105cells / well in 12-well plates up to 24 hours before transfection. siRNA transfection complexes were formed in DharmaFECT 1 reagent (Horizon Discovery T-2001) at 1 pL:10 nM final siRNA ratio in OPTI-MEM™ (Gibco 31985062) following suggested manufacturer protocols. Cells were transfected with 25 nM final concentration of siRNA. Cells were incubated 48 hours before downstream analysis.RNA extraction, reverse transcription, and quantitative real-time PCR (qRT-PCR)

[0168] RNA was isolated by RNEASY® Plus Mini Kit (QIAGEN® Group, Hilden, Germany, Catalogue no 74134) following manufacturer protocol, with modification for total RNA isolation. RNA was quantified by QUBIT® RNA Broad Range assay (INVITROGEN® Q32852). cDNA was generated from 100 ng total RNA using the SUPERSCRIPT® IV kit (INVITROGEN® 18091050) and random hexamer priming. RNA reactions without RT were carried throughout subsequent reactions as controls. qPCR was performed using TAQMAN® Fast Universal PCR Master Mix and the associated protocol (Applied Biosystems 4444964). TAQMAN® probes for GGTA1 (ThermoFisher, Assay ID:SsO3388O86_sl) and custom designed assays for SDHA(F: 5’ACCGCACTGGCCACTCA-3’ SEQ ID NO: 96; R: 5’AAATACTCCACAAAATAGCTGGTATCG-3’ SEQ ID NO: 97; probe: 5’TGTACGGAAGGTCTCTG-3’ SEQ ID NO: 98) and CD163 (F:5’AGGATGCTGGAGTGATTTGCTT-3’ SEQ ID NO: 99; R:5’TCAGTGACTCCATCTACCACTCTCA-3’ SEQ ID NO: 100; probe:5’AATGGAGCAGACCTGAAA-3’ SEQ ID NO: 101) (IDT) were used. Relative differential gene expression was calculated using the 2'AACtMethod (Livak, K. J. and Schmittgen, T.D., Methods, 2001, 25, 402-408.) using SDHA as a reference gene and untransfected parental cell types as the reference sample.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)EXAMPLE 1

[0169] This example illustrates selection of potential GEiGS® sites for gene silencing of CD163.

[0170] For the desired cell type, porcine pulmonary alveolar macrophages (“CPAMs”), for CD 163, small RNA sequencing was performed. Highly expressed miRNAs were screened for potential near reverse complement matches to the target gene, and 50 candidate miRNAs were selected for in vitro screening. The selected miRNAs are listed in Table 2.EXAMPLE 2

[0171] This example illustrates silencing of CD 163 in CPAM cells.

[0172] Mature GEiGS® solutions targeting CD 163 were ordered as synthetic siRNAs (SEQ ID NOs: 51-95, see Table 2) and transfected into hTERT-immortalized porcine pulmonary alveolar macrophages (CPAMs), in accordance with the methods disclosed herein and as described below. Cells were transfected with 25 nM final concentration of siRNA. Table 5 shows the silencing efficiency of the siRNAs, as measured by qPCR of the CD 163 transcript. Numbers for each solution correspond to the first column of Table 2. Dummy sequences are non-silencing equivalents for each siRNA. Differential gene expression was calculated as described below, using succinate dehydrogenase complex flavoprotein subunit A (SDHA) as the reference gene. Statistical significance (p-value) was calculated by ordinary one-way ANOVA with multiple comparisons between untransfected CPAM controls and solutions.Table 5<Atty. Dkt. No. 102726-0300(TD-20-2024-W01)><<>>>Atty. Dkt. No. 102726-0300(TD-20-2024-W01)><>Atty. Dkt. No. 102726-0300(TD-20-2024-W01)<>>>>>><>The samples designated as “soln_01,” “soln_02,” etc., as shown in Table 5, correspond to the predicted mature sRNA molecules as set forth in SEQ ID NOs: 51-95 as shown in column 6 of Table 2. The number following each solution (“soln”) corresponds to the number in the first column of Table 2. The samples designated as “dummy 01,” “dummy _02,” etc., as shown in Table 5, correspond to the predicted mature dummy sRNA molecules as set forth in SEQ ID NOs: 210-224 as shown in column 6 of Table 4.

[0173] This example demonstrates that GEiGS® solutions of the present teachings are capable of knockdown of CD 163 expression in CPAM cells. Accordingly, these results demonstrate that the compositions and methods of the present technology are useful forAtty. Dkt. No. 102726-0300(TD-20-2024-W01)preventing PRRSv infection in pigs and for generating gene-edited pigs having resistance to PRRSv.EXAMPLE 3

[0174] This example illustrates a method of testing potential editing reagents for use in creating GEiGS® solutions in vitro.

[0175] GEiGS® sites were selected based on the knock-down efficiency in EXAMPLE 2 and the phenotypic data from EXAMPLE 5. sgRNAs and HDR templates (as shown in Table 3) were designed to edit the genomic sites (miR scaffolds) described above (see Table 2) to form genes encoding GEiGS® precursor miRNA in the porcine genome. To test the editing efficiency of the sgRNAs and HDR templates in vitro, editing reagents were nucleofected into porcine embryonic stem cells (ESCs) derived from wildtype blastocysts. Then, 20 picomole (pmol) of Cas9 protein and of in vitro synthesized single guide RNA was combined in water to a total volume of 2.5 pl. The RNPs were combined with 25 pmol HDR template per reaction prior to nucleofection. In preparation for nucleofection, ESCs were harvested using Accutase®, culture medium was removed from cells, washed with Dulbecco’s phosphate buffered saline (DPBS), and incubated for 5-8 minutes at 38.5°C in the presence of Accutase®. Cells were harvested with complete medium and pelleted via centrifugation (300 g x 3 min at room temperature). The supernatant was discarded, and cells were resuspended in an appropriate volume of DPBS to obtain a single cell suspension determined by cell count using trypan blue staining.

[0176] Cells were then resuspended in nucleofection buffer P3 at a final concentration of approximately 20,000 cells per 20 pl, with 20 pl of the cell suspension pipetted into each well of a nucleofection plate containing the RNP mixture, and mixed gently to resuspend the cells. The RNP / cell mixture was transferred in the nucleofection plate and nucleofected with program DS137 on a Lonza Bioscience® electroporator. Then, 80 pl of warm ABI media consisting of Advanced DMEMZF12 (Dulbecco’s Modified Eagle Medium / Ham’s F-12), 1% GlutaMAX (ThermoFisher SCIENTIFIC), O.lpl / mL IWR-l-endo (Wnt antagonist, Calbiochem®), 15ng / mL Recombinant Human basic Fibroblast Growth Factor (bFGF), 0.2 pl / mL TGF-beta 1 protein (TGF-B1), and 0.75% AlbumiNZ™ ovine serum albumin (BSA)Atty. Dkt. No. 102726-0300(TD-20-2024-W01)was added to each well. The suspensions were mixed gently by pipetting, then 100 pl was transferred to a 12- well plate containing 900 pl of ABI media pre-incubated at 38.5°C. The plate was then incubated at 38.5°C, 5% CO2, and 5% O2 until confluency was observed, or approximately 48 to 96 hours. Following nucleofection and incubation, genomic DNA was prepared from transfected and control ESCs by adding 15 pl of QuickExtract® DNA Extraction Solution to pelleted cells, lysed by incubation for 10 minutes at 37°C, for eight minutes at 65°C, for five minutes at 95°C, after which lysate could be was held at -20°C until used for DNA sequencing.EXAMPLE 4

[0177] This example illustrates a method of silencing CD163 in ESCs differentiated into macrophage cells (ESC-MACs) carrying candidate GEiGS® solution sequences. Numbers for each solution correspond to the first column of Table 2.

[0178] Candidate GEiGS® solution sequences were integrated into a native genomic locus in porcine embryonic stem cells (ESCs) as described in EXAMPLE 3. Integration of the GEiGS® solutions was validated by Next-Generation Sequencing.

[0179] The ESCs were then differentiated into ESC-MACs, which natively express CD163, using methods known to those of skill in the art. The silencing efficiency of the candidate GEiGS® solution sequences was measured by qPCR of the CD 163 transcript in the ESC-MACs. Table 6 shows the differential gene expression (DGE) of CD 163 in the ESC-MACs containing candidate GEiGS® solution sequences, as well as in unedited, wild-type ESC-MACs used as a positive control. DGE was calculated as described supra, using succinate dehydrogenase complex flavoprotein subunit A (SDHA) as the reference gene.Table 6Atty. Dkt. No. 102726-0300(TD-20-2024-W01)The samples designated as “soln_08,” “soln_09,”“soln_10,” and “soln_40” as shown in Table 6,correspond to the GEiGS® solution sequences as setforth in SEQ ID NOs: 8, 9, 10, and 40, respectivelyas shown in column 5 of Table 2. The numberfollowing each solution (“soln”) corresponds to thenumber in the first column of Table 2.

[0180] This example demonstrates that GEiGS® solutions of the present teachings are capable of silencing CD 163 expression in ESC-MACs. Accordingly, these results demonstrate that the compositions and methods of the present technology are useful for prevention of PRRSv infection and for the generation of gene-edited pigs having resistance to PRRSv.EXAMPLE 5

[0181] This example illustrates a method of testing viral resistance in cells carrying candidate GEiGS® solution sequences.

[0182] Candidate GEiGS® solution sequences were integrated into a native genomic locus in porcine embryonic stem cells (ESCs) as described in EXAMPLE 3. Numbers for each solution correspond to the first column of Table 2. Integration of the GEiGS® solutions was validated by Next-Generation Sequencing.

[0183] The ESCs were then differentiated into ESC-MACs, which natively express CD163, using methods known to those of skill in the art. The cells were serially infected with dilutions of PRRS virus type from log-1 to through log-7 in order to calculate the 50% tissue culture infectious dose (TCID50) / mL, and then normalized to the WT, alongside control (unedited wild type) cells. Stock virus was at a concentration of 8.93E+07Atty. Dkt. No. 102726-0300(TD-20-2024-W01)TCID50 / mL based on infection of WT unedited cells. After 1-2 days, cells were evaluated for PRRS virus infection by immunostaining for the presence of the PRRSV N-protein. Table 7 demonstrates that the GEiGS®- containing cells show reduced infection and / or replication of PRRSv relative to the wild type cells as measured by TCID50 / ml, which was calculated utilizing the Spearman- Karber method (Lei Chengfeng, etal., On the Calculation of TCIDso for Quantitation of Virus Infectivity, Virologica Sinica, 36(1): 141-144 (2020)). TCID50 is a measure of viral infectivity as determined from the dilution at which the inoculum infects half of a target culture.Table 7The samples designated as “soln_08,” “soln_09,” “soln_10,” and“soln_40” as shown in Table 7, correspond to the GEiGS® solutionsequences as set forth in SEQ ID NOs: 8, 9, 10, and 40, respectively asshown in column 5 of Table 2. The number following each solution(“soln”) corresponds to the number in the first column of Table 2.

[0184] This example demonstrates that the GEiGS® knockdown of CD 163 results in increased resistance of ESC-MAC cells to PRRS virus type 1 and / or PRRS virus type 2. Accordingly, these results demonstrate that the compositions and methods of the present technology are useful for preventing PRRSv infection in pigs and for generating gene-edited pigs having resistance to PRRSv.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)EXAMPLE 6

[0185] This example illustrates a potential method of in vivo testing of editing reagents for GEiGS® creation in porcine embryos.

[0186] Porcine oocytes can be obtained and then matured and fertilized in vitro using methods known in the art. Fertilized zygotes are subjected to a single 2 picoliter (pl) cytoplasmic injection of Cas9 RNP complex plus HDR template as described in EXAMPLE 4 and cultured to the blastocyst stage using standard methods.

[0187] 7 day blastocysts can be harvested and the DNA isolated for next generation sequencing to confirm the presence of the desired edit. The editing reagents providing the best desired editing frequency will be selected to make gene-edited pigs.EXAMPLE 7

[0188] This example illustrates the creation of gene-edited pigs.

[0189] To prepare embryo donors, pubertal gilts from PIC™ (Pig Improvement Company, Basingstoke, UK) lines can be subjected to estrus synchronization by treatment with the progesterone analog Regumate (24-36 mg / animal) for 14 days. Oocytes can be obtained by standard methods known in the art and fertilized in vitro using semen that can be obtained from the corresponding male line. RNP complexes can be prepared as in EXAMPLE 4 and injected into the elite zygotes as in EXAMPLE 6. The injected zygotes can then cultured to the blastocyst stage and implanted into surrogate females according to methods known in the art.EXAMPLE 8

[0190] This example illustrates molecular characterization of pigs generated in EXAMPLE 7.

[0191] A tissue sample can be taken from a pig edited according to EXAMPLE 7. Suitable tissue sources include, but are not limited to, tail, ear notch, or blood samples. The tissueAtty. Dkt. No. 102726-0300(TD-20-2024-W01)sample can be frozen at -20°C within 1 hour of sampling to preserve integrity of the DNA in the tissue sample.

[0192] DNA can be extracted from tissue samples after proteinase K digestion in lysis buffer. Characterization can be performed on two different sequence platforms, short sequence reads using the ILLUMINA® platform (ILLUMINA®, Inc., San Deigo, CA) and long sequence reads on an Oxford NANOPORE™ platform (Oxford NANOPORE™ Technologies, Oxford, UK).

[0193] For short sequence reads, two-step PCR can be used to amplify and sequence the region of interest. The first step can be a locus-specific PCR which amplifies the locus of interest from the DNA sample using a combined locus-specific primer with a vendorspecific primer. The second step can attach the sequencing index and adaptor sequences to the amplicon from the first step so that sequencing can occur.

[0194] The locus-specific primers for the first step PCR can be chosen so that they amplify a region <300bp such that ILLUMINA® paired-end sequencing reads can span the amplified fragment. Multiple amplicons are preferred to provide redundancy should deletions or point mutations prevent primers from correctly binding. Sequence data for the amplicon can be generated using an ILLUMINA® sequencing platform (e.g., MISEQ™, ILLUMINA®, Inc., San Deigo, CA). Sequence reads are analyzed to characterize the outcome of the editing process.

[0195] For long sequence reads, two-step PCR can be used to amplify and sequence the region of interest. The first step can be a locus-specific PCR, which amplifies the locus of interest from the DNA sample using a combined locus-specific primer with a vendorspecific adapter. The second step PCR attaches the sequencing index to the amplicon from the first-step PCR so the DNA can be ready for preparing a sequencing library. The step 2 PCR products undergo a set of chemical reactions from a vendor kit to polish the ends of the DNA and ligate on the adapter containing the motor protein to allow access to the pores for DNA strand-based sequencing.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)

[0196] The locus-specific primers for the first step PCR range can be designed to amplify different regions of the porcine genome and amplified regions differing in length.Normalized DNA can then be mixed with vendor-supplied loading buffer and loaded onto the NANOPORE™ flowcell.

[0197] Long sequence reads, while having lower per base accuracy than short reads, are very useful for observing the long-range context of the sequence around the target site.

[0198] This example will illustrate confirmation of the desired gene edit in pigs.EXAMPLE 9

[0199] This example illustrates confirmation of knockdown of CD 163 in gene-edited pigs.

[0200] For CD163 GEiGS® edited pigs, tissue samples of PAM cells can be obtained by bronchoalveolar lavage. Tissue can be lysed and then RNA isolated and sequenced as described above. Knockdown of the gene of interest can be confirmed.EXAMPLE 10

[0201] This example illustrates a potential method of PRRSv challenge of pigs carrying GEiGS® sequences directed to CD 163.

[0202] Pigs carrying GEiGS® sequences directed to CD 163 as created in EXAMPLE 7 can be inoculated with 3 ml of PRRSv Type II (NVSL 97-7895) having 104 to 105 virions (4-5 log TCIDso). 1.5 ml can be administered intramuscularly with a 21 gauge needle. The remaining 1.5 ml can be administered intranasally. Serum samples can be obtained on Day 0 (prior to inoculation on that day), Day 3, Day 5, Day 7, Day 10, Day 14, and Day 21.Realtime PCR can be used to determine the presence of virus in the serum samples using TETRACORE® EZ-PRRSV MPX 4.0 Master Mix and Enzyme with rifamycin monooxygenases (ROX) according to manufacturer directions. It is expected that pigs showing CD 163 knockdown in PAM cells will show reduced presence of virus in the serum compared to wild type pigs.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)EQUIVALENTS

[0203] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0204] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0205] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2,Atty. Dkt. No. 102726-0300(TD-20-2024-W01)or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0206] All publicly available documents referenced or cited herein, such as patents, patent applications, provisional applications, and publications, including GenBank Accession Numbers, are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0207] Other embodiments are set forth within the following claims.

Claims

Atty. Dkt. No. 102726-0300(TD-20-2024-W01)CLAIMSWhat is claimed is:

1. A porcine animal or porcine cell comprising one or more gene-edited chromosomal sequences, each encoding a non-coding RNA molecule that silences an endogenous CD 163 gene.

2. The porcine animal or porcine cell according to claim 1, wherein the non-coding RNA molecule encodes or is processed into an siRNA having a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 51-95.

3. The porcine animal or porcine cell according to claim 1 or claim 2, wherein the noncoding RNA molecule decreases the expression or activity of CD 163 protein as compared to the expression or activity in a porcine animal or porcine cell that lacks the one or more gene-edited chromosomal sequences.

4. The porcine animal or porcine cell according to any one of claims 1-3, wherein the porcine animal or porcine cell is resistant to Porcine Reproductive and Respiratory Syndrome virus (PRRSv).

5. The porcine animal or porcine cell according to any one of claims 1-4, wherein the one or more gene-edited chromosomal sequences encoding the non-coding RNA molecule reduces susceptibility of the porcine animal or porcine cell to infection by PRRSv.

6. The porcine animal or porcine cell according to any one of claims 1-5, wherein the one or more gene-edited chromosomal sequences comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-50.

7. The porcine animal or porcine cell according to any one of claims 1-6, wherein the non-coding RNA molecule is only expressed in pulmonary alveolar macrophages.

8. The porcine animal or porcine cell according to claim 1, wherein the one or more gene edited-chromosomal sequences comprise an edit of an endogenous non-codingAtty. Dkt. No. 102726-0300(TD-20-2024-W01)RNA molecule that redirects silencing activity of the encoded non-coding RNA molecule to CD 163.

9. The porcine animal or porcine cell according to claim 8, wherein the non-coding RNA molecule encodes or is processed into an siRNA having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 51-95.

10. The porcine animal or porcine cell according to claim 8 or 9, wherein the one or more gene-edited chromosomal sequences comprise a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-50.

11. The porcine animal or porcine cell according to claim 10, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of:a. SEQ ID NO: 8;b. SEQ ID NO: 9;c. SEQ ID NO: 10;d. SEQ ID NO: 40;e. SEQ ID NOs: 8 and 10;f. SEQ ID NOs: 9 and 10;g. SEQ ID NOs: 9 and 40; andh. SEQ ID NOs: 10 and 40.

12. The porcine animal or porcine cell according to claim 11, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of:a. SEQ ID NO: 8;b. SEQ ID NO: 9;c. SEQ ID NO: 40;d. SEQ ID NOs: 8 and 10;e. SEQ ID NOs: 9 and 40; andf. SEQ ID NOs: 10 and 40.

13. The porcine animal or porcine cell according to claim 11, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of:Atty. Dkt. No. 102726-0300(TD-20-2024-W01)a. SEQ ID NO: 8;b. SEQ ID NO: 9;c. SEQ ID NO: 10;d. SEQ IDNOs: 9 and 10;e. SEQ ID NOs: 9 and 40; andf. SEQ IDNOs: 10 and 40.

14. The porcine animal according to claim 1, wherein the porcine animal is a gene-edited Pig-15. An isolated porcine cell obtained from the porcine animal of any one of claims 1-14.

16. The isolated porcine cell of claim 15, wherein the cell is selected from a germ cell, a somatic cell, a primary cell, a stem cell, an embryonic stem cell, an adult stem cell, a hematopoietic stem cell, a mesenchymal stem cell, a gamete cell, a zygote cell, a blastocyst cell, an embryo, or a fetus.

17. The isolated cell of claim 16, wherein the germ cell is a spermatogonia or an oogonia.

18. The isolated cell of claim 16, wherein the gamete cell is a sperm cell or an oocyte.

19. An siRNA comprising any one of SEQ ID NOs: 51-95.

20. An miRNA comprising any one of SEQ ID NOs: 1-50.

21. A method for producing a porcine animal or porcine cell comprising one or more gene-edited chromosomal sequences, each encoding a non-coding RNA molecule that silences an endogenous CD 163 gene, the method comprising contacting the porcine animal or porcine cell with an effective amount of a DNA editing agent, wherein the DNA editing agent is configured to edit a chromosomal sequence to produce one or more gene-edited chromosomal sequences, each encoding a noncoding RNA molecule that silences an endogenous CD 163 gene, thereby producing a porcine animal or porcine cell having decreased expression or activity of CD 163 protein as compared to the expression or activity of CD 163 in a porcine animal or porcine cell that lacks the edited chromosomal sequence.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)22. The method of claim 21, wherein the DNA editing agent comprises at least one guide RNA (gRNA) operably linked to a constitutive or inducible promoter.

23. The method of claim 21 or 22, wherein the DNA editing agent comprises an endonuclease.

24. The method of any one of claims 21-23, wherein the DNA editing agent comprises a meganuclease, a zinc finger nuclease, a transcription-activator like effector nuclease, or CRISPR.

25. The method of any one of claims 21-24, wherein the non-coding RNA molecule is produced via homologous DNA repair (HDR) with a donor template sequence comprising a modification of interest.

26. The method of any one of claims 21-25, wherein the non-coding RNA molecule encodes or is processed into a siRNA.

27. The method of claim 26, wherein the siRNA is selected from the group consisting of:SEQ IDNOs: 51-95.

28. The method of claim 21 or 27, wherein the one or more gene-edited chromosomal sequences comprise a nucleic acid sequence selected from the group consisting of: SEQ IDNOs: 1-50.

29. The method of claim 28, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of:a. SEQ IDNO: 8;b. SEQ IDNO: 9;c. SEQ IDNO: 10;d. SEQ IDNO: 40;e. SEQ IDNOs: 8 and 10;f. SEQ IDNOs: 9 and 10;g. SEQ ID NOs: 9 and 40; andh. SEQ IDNOs: 10 and 40.Atty. Dkt. No. 102726-0300(TD-20-2024-W01)30. The porcine animal or porcine cell according to claim 29, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of: a. SEQ ID NO: 8;b. SEQ ID NO: 9;c. SEQ ID NO: 40;d. SEQ IDNOs: 8 and 10;e. SEQ ID NOs: 9 and 40; andf. SEQ IDNOs: 10 and 40.

31. The porcine animal or porcine cell according to claim 29, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of: a. SEQ ID NO: 8;b. SEQ ID NO: 9;c. SEQ ID NO: 10;d. SEQ IDNOs: 9 and 10;e. SEQ ID NOs: 9 and 40; andf. SEQ IDNOs: 10 and 40.

32. A method for producing a porcine animal or porcine cell resistant to Porcine Reproductive and Respiratory Syndrome virus (PRRSv), the method comprising contacting the porcine animal or porcine cell with an effective amount of a DNA editing agent,wherein the DNA editing agent is configured to edit a chromosomal sequence to produce one or more gene-edited chromosomal sequences, each encoding a noncoding RNA molecule that silences an endogenous CD 163 gene, thereby producing a porcine animal or porcine cell having decreased expression or activity of CD 163 protein as compared to the expression or activity of CD 163 in a porcine animal or porcine cell that lacks the edited chromosomal sequence.

33. The method of claim 32, wherein when the porcine animal or porcine cell is infected with PRRSv, the animal or cell has an increased resistance to PRRSv infectivity asAtty. Dkt. No. 102726-0300(TD-20-2024-W01)compared to an infected porcine animal or porcine cell that lacks the one or more gene-edited chromosomal sequences.

34. The method of claim 32 or 33, wherein the DNA editing agent comprises at least one guide RNA (gRNA) operably linked to a constitutive or inducible promoter.

35. The method of any one of claims 32-34, wherein the DNA editing agent comprises an endonuclease.

36. The method of any one of claims 32-35, wherein the DNA editing agent comprises a meganuclease, a zinc finger nuclease, a transcription-activator like effector nuclease, or CRISPR.

37. The method of any one of claims 32-36, wherein the non-coding RNA molecule is produced via homologous DNA repair (HDR) with a donor template sequence comprising a modification of interest.

38. The method of any one of claims 32-37, wherein the non-coding RNA molecule encodes or is processed into a siRNA.

39. The method of claim 38, wherein the siRNA is selected from the group consisting of:SEQ IDNOs: 51-95.

40. The method of any one of claims 32-39, wherein the one or more gene-edited chromosomal sequences comprise a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-50.

41. The method of claim 40, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of:a. SEQ ID NO: 8;b. SEQ ID NO: 9;c. SEQ ID NO: 10;d. SEQ ID NO: 40;e. SEQ IDNOs: 8 and 10;f. SEQ IDNOs: 9 and 10;Atty. Dkt. No. 102726-0300(TD-20-2024-W01)g. SEQ ID NOs: 9 and 40; andh. SEQ ID NOs: 10 and 40.

42. The porcine animal or porcine cell according to claim 41, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of: a. SEQ ID NO: 8;b. SEQ ID NO: 9;c. SEQ ID NO: 40;d. SEQ ID NOs: 8 and 10;e. SEQ ID NOs: 9 and 40; andf. SEQ ID NOs: 10 and 40.

43. The porcine animal or porcine cell according to claim 41, wherein the one or more gene-edited chromosomal sequences are selected from the group consisting of: a. SEQ ID NO: 8;b. SEQ ID NO: 9;c. SEQ ID NO: 10;d. SEQ ID NOs: 9 and 10;e. SEQ ID NOs: 9 and 40; andf. SEQ ID NOs: 10 and 40.

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