Recombinant aav production through avian aav pseudotyping

Abstract

The present disclosure provides engineered nucleic acids and compositions thereof that may be used to produce pseudotyped avian rAAV particles. Additionally, the present disclosure provides methods and kits comprising engineered nucleic acids for pseudotyped avian rAAV particle manufacturing. Moreover, the present disclosure provides suitable manufacturing systems. Accordingly, the pseudotyped avian rAAV particles of the disclosure can be isolated and purified at high viral titers that cannot be achieved with conventional rAAV particle production approaches. The pseudotyped avian rAAV particles of the present disclosure are also more suitable for administration in a clinical setting than rAAV particles previously described in the art as they are less immunogenic and, therefore, more compatible for in vivo applications.

Description

[0001] RECOMBINANT AAV PRODUCTION THROUGH AVIAN AAV PSEUDOTYPING

[0002] RELATED APPLICATIONS

[0003] The application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application number 63 / 514,319 filed July 18, 2023, which is incorporated by reference in its entirety.

[0004] FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant number R01 HD052682, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (U120270134WO00-SEQ.xml; Size: 160,499 bytes; and Date of Creation: June 18, 2024) is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Recombinant adeno-associated virus (rAAV) particles are a gene therapy delivery system for research and clinical applications. Cost-effective and scalable methods for rAAV production with high yields and purity levels of rAAV particles are important for advancing life science research and medicine.

[0010] SUMMARY

[0011] Aspects of the present disclosure relate to nucleic acids, manufacturing systems, and methods for producing pseudotyped avian rAAV particles.

[0012] In some aspects, the present disclosure provides a plurality of nucleic acids comprising a first nucleic acid comprising a heterologous nucleic acid flanked by a first and second avian adeno-associated virus (AAV) inverted terminal repeat (ITR), and a second nucleic acid comprising at least one gene sequence encoding an AAV capsid protein (e.g., a mammalian AAV capsid protein, for example a human AAV capsid protein, a bovine AAV capsid protein, or other mammalian AAV capsid protein) and at least one gene sequence encoding an avian AAV rep protein. In some embodiments, the heterologous nucleic acid comprises a gene sequence encoding an RNA. In some embodiments, the gene sequence is operably linked to at least one regulatory sequence. In some embodiments, the at least one regulatory sequence is selected from the group consisting of a promoter, an enhancer, a poly(A) signal, an intron, a Woodchuck Hepatitis Virus Post-Transcriptional Response Element (WPRE), a splicing donor / splicing acceptor element, and a bipartite leader (BPL) sequence comprising a Kozak sequence. In some embodiments, the promoter is a CMV promoter. In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the poly(A) signal is a Proudfoot poly(A) signal. In some embodiments, the heterologous nucleic acid comprises from 5’ to 3’, in relative order, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, the gene sequence encoding an RNA, and a Proudfoot poly(A) signal. In some embodiments, the gene sequence encodes a therapeutic RNA or therapeutic protein. In some embodiments, the therapeutic protein is therapeutic for a disease, disorder, or condition described herein. In some embodiments, the therapeutic RNA is selected from the group consisting of an interfering RNA, exon- skipping RNA, enzymatic RNA, guide RNA, small nuclear RNA, ribosomal RNA, and transfer RNA, wherein the therapeutic protein is selected from the group consisting of enzyme, enzymatic domain, enzyme substrate, hormone, receptor, gene editor, peptibody, growth factor, clotting factor, cytokine, chemokine, ion channel-activating or inhibitory peptide, cell-permeable peptide, thrombolytic, bone morphogenetic protein, Fc-fusion protein, anticoagulant, and antibody or antigen-binding fragment thereof.

[0013] In some embodiments, the heterologous nucleic acid comprises a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 25-29. In some embodiments, the heterologous nucleic acid comprises the sequence of any one of SEQ ID NOs: 25-29.

[0014] In some embodiments, the first nucleic acid is a vector comprising from 5’ to 3’, in relative order, a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, the first avian AAV ITR, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, the therapeutic gene sequence, and a Proudfoot poly(A) signal, the second avian AAV ITR, and a bovine growth hormone (bGH) poly(A) signal. In some embodiments, the vector further comprises a 5’ chicken embryo lethal orphan (CELO) virus ITR and a 3’ CELO virus ITR. In some embodiments, the vector comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 23-31. In some embodiments, the vector comprises a nucleic acid sequence of any one of SEQ ID NOs: 23-31. In some embodiments, the second nucleic acid is a vector comprising from 5’ to 3’, in relative order, a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, the at least one gene sequence encoding an avian rep protein, the at least one gene sequence encoding an AAV capsid protein, and a bovine growth hormone (bGH) poly(A) signal. In some embodiments, the vector further comprises a 5’ CELO virus ITR and a 3’ CELO virus ITR. In some embodiments, the at least one AAV capsid protein is selected from the group consisting of a mammalian AAV1, mammalian AAV5, mammalian AAV6, mammalian AAV7 mammalian AAV9, and mammalian AAV8-M3. In some embodiments, the second nucleic acid comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 1-20 or 32-33. In some embodiments, the second engineered nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-20 or 32-33.

[0015] In some embodiments, the plurality of nucleic acids further comprises a helper nucleic acid. In some embodiments, the helper nucleic acid comprises at least one of El, E2A, E4, or VA genes. In some embodiments, the helper nucleic acid is provided on a third nucleic acid. In some embodiments, the third nucleic acid is a vector. In some embodiments, the helper nucleic acid is an adenovirus nucleic acid. In some embodiments, the helper nucleic acid is an avian adenovirus nucleic acid. In some embodiments, the helper nucleic acid is a CELO helper virus nucleic acid. In some embodiments, the helper nucleic acid comprises a sequence with least 75% (e.g., 75-80%, 80-85%, 85-90%, or 95-99%) identity to a nucleotide sequence comprised in SEQ ID NO: 36 (e.g., at least 75% identity to a helper sequence comprised in the nucleotide sequence set forth in SEQ ID NO: 36, such as a helper sequence comprising at least one of El, E2A, E4, or VA genes).

[0016] Other aspects of the present disclosure relate to manufacturing systems comprising the plurality of nucleic acids. In some embodiments, the manufacturing system is an avian egg or a host cell. In some embodiments, the avian egg is selected from the group consisting of chicken egg, duck egg, goose egg, and quail egg. In some embodiments, the avian egg is an embryonated egg. In some embodiments, the host cell is a cell of a cell line. In some embodiments, the host cell is an avian cell. In some embodiments, the avian cell is selected from the group consisting of chicken cell, duck cell, goose cell, and quail cell. In some embodiments, the chicken cell is a EB14 cell, HD- 11 cell, DF-1 cell, DT95 cell, DT40 cell, LMH cell, or PBS- 12 SF cell. In some embodiments, the duck cell is an AGE1.CR cell, AGEl.CR.pIX cell, EB66 cell, or DEF cell. In some embodiments, the quail cell is a QEF cell, a QM7 cell, a QT-35 cell, or a QT-6 cell. In some embodiments, the host cell is a suspension cell. In some embodiments, the cell is a HEK293 cell. In some embodiments, the manufacturing system is a cell population comprising a plurality of the host cells.

[0017] In some embodiments, a manufacturing system further comprises a helper nucleic acid (e.g., a cell line stably transfected with a helper nucleic acid or a manufacturing system that has been contacted with a helper nucleic acid). In some embodiments, the helper nucleic acid is an adenovirus nucleic acid. In some embodiments, the helper nucleic acid is an avian adenovirus nucleic acid. In some embodiments, the helper nucleic acid is a CELO helper virus nucleic acid. In some embodiments, the helper nucleic acid comprises a sequence with least 75% identity to a nucleotide sequence comprised in SEQ ID NO: 36 (e.g., at 75% identity to a helper sequence comprised in the nucleotide sequence set forth in SEQ ID NO: 36, such as a helper sequence comprising at least one of El, E2A, E4, or VA genes).

[0018] Other aspects of the present disclosure relate to methods of producing an rAAV particle. In some embodiments, methods of producing an rAAV particle comprise contacting a manufacturing system with the plurality of nucleic acids, incubating the manufacturing system, and isolating the rAAV particle from the manufacturing system.

[0019] In some embodiments, contacting comprises inoculating the avian egg. In some embodiments, an allantoic cavity, a chorioallantoic membrane, a yolk sac, or an amnion of the avian egg is inoculated. In some embodiments, inoculation comprises manual injection. In some embodiments, inoculation comprises automated injection (e.g., automated injection performed in a GMP setting). In some embodiments, the avian egg is incubated in an automatic egg incubator. In some embodiments, the avian egg is incubated after inoculation for a period of at least 15 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 80 hours, at least 90 hours, or at least 96 hours.

[0020] In some embodiments, the isolated rAAV particle is substantially free of avian viruses. In some embodiments, the isolated rAAV particle is isolated from the allantoic fluid. In some embodiments, the isolated rAAV particle is isolated using a manual pipette, a manual syringe, a machine-controlled pipette, or a machine-controlled syringe.

[0021] In some embodiments, methods of producing an rAAV particle further comprises a purification step. In some embodiments, the isolated rAAV particle is subjected to iodixanol gradient ultracentrifugation and / or contacted with an affinity resin.

[0022] In some embodiments, the isolated rAAV particle or purified rAAV particle is at a titer of at least about 1 x 108, at least about 5 x 108, at least about 1 x 109, at least about 5 x 109, at least about 1 x IO10, at least about 5 x IO10, at least about 1 x 1011, at least 2 x 1011, or at least 5 x 1011vector genomes (vg) / ml. Other aspects of the present disclosure relate to an rAAV particle comprising the first nucleic acid of the plurality of nucleic acids and at least one AAV capsid protein (e.g., a mammalian AAV capsid protein, for example a human AAV capsid protein, a bovine AAV capsid protein, or other mammalian AAV capsid protein). In some embodiments, at least one AAV capsid protein comprises a mammalian AAV1, mammalian AAV5, mammalian AAV6, mammalian AAV7 mammalian AAV9, and mammalian AAV8-M3.

[0023] Other aspects of the present disclosure relate to compositions (e.g., pharmaceutical compositions) comprising rAAV particles, such as compositions produced by methods described herein. In some aspects, rAAV particles provided herein, and pharmaceutical compositions thereof, may be used in method of treating a subject. In some embodiments, the suspect has a disease, disorder, or condition described herein.

[0024] BRIEF DESCRIPTION OF DRAWINGS

[0025] FIGs. 1A-1B show schematics of examples of avian AAV, rAAV genomes, and nucleic acids for rAAV production. FIG. 1A shows a non-limiting example of an avian AAV genome comprising gene sequences encoding avian AAV rep and avian AAV capsid proteins flanked by avian AAV inverted terminal repeats (ITRs) and an AAV particle (also known as a virion) comprising the same. FIG. IB shows a non-limiting schematic of an example of an avian rAAV genome comprising a heterologous nucleic acid flanked by avian AAV ITRs. \

[0026] FIGs. 2A-2Care non-limiting schematics of nucleic acids for rAAV production and AAV packaging methods of the present disclosure. FIG. 2A shows a triple transfection protocol. FIG. 2B shows a protocol for generating a stable host cell expressing rAAV packaging and helper nucleic acids which may be subsequently transfected with a recombinant viral genome comprising a transgene. FIG. 2C shows a triple transfection-inoculation protocol wherein a plurality of vectors (providing the rAAV genome and packaging and helper nucleic acids) are transfected into an embryonated egg.

[0027] FIGs. 3A-3B show non-limiting schematics depicting examples of constructs used for production of pseudotyped avian rAAV particles. FIG. 3A shows a schematic of an example of an avian rAAV genome including a heterologous nucleic acid comprising a transgene which is flanked by avian AAV ITRs. FIG. 3B shows a schematic of an example nucleic acid comprising a gene sequence encoding an avian AAV rep protein and a gene sequence encoding an AAV capsid protein that may function as a packaging nucleic acid to help produce a virus particle (also known as a virion). FIG. 4 shows an example of a transfection strategy for production of pseudotyped avian rAAV particles in mammalian (e.g., HEK293T) and avian (e.g., QT6) cells that are capable of reinfecting target cells.

[0028] FIG. 5 shows a schematic of a non-limiting example of an AAV packaging method in embryonated avian eggs. A transfection-inoculation protocol is shown, as well as downstream harvesting and purification steps.

[0029] FIGs 6A-6B show diagrams that illustrate the anatomy of an embryonated avian egg. FIG. 6A shows the chalazae, yolk, blastodisc, egg white, airspace, inner shell membrane, outer shell membrane, shell, and cuticle. FIG. 6B shows various routes of injection of engineered nucleic acids and / or vectors into the egg’s chorioallantoic membrane (CAM) in order to inoculate the allantoic fluid (allantoic cavity).

[0030] FIGs. 7A-7C show non-limiting examples of steps of egg inoculation and harvesting. FIG. 7A shows an example of a method wherein, prior to inoculation, embryonated eggs are candled to determine their stage of development. FIG. 7B shows an example method for inoculating eggs with AAV vectors by piercing through the shell and CAM using a 20 gauge, 1.5-inch syringe attached to an egg-piercing rubber stopped. FIG. 7C shows an example method for egg harvesting wherein, once the embryo is determined to be no longer viable by candling, the egg is opened and sterile scissors are used to cut away the shell around the air sac. Allantoic fluid is aspirated using a syringe or a pipette.

[0031] FIGs. 8A-8C show images of examples of high-throughput automatic egg injectors having a production capacity of 62,000 eggs / hour. FIG. 8A shows a photograph of an exemplary automatic egg injector piercing the shell of an embryonated egg. FIG. 8B depicts piercing of the CAM and inoculation of the allantoic fluid by an automatic injector. FIG. 8C shows a photograph of a multi-channel automatic egg injection system.

[0032] FIG. 9 shows a step-by-step diagram of images showing allantoic cavity harvesting after inoculation with rAAV. The egg is opened by tapping on the shell just above the air sac, and sterile scissors are used to cut away the shell around the air sac and cut through the egg’s CAM.

[0033] FIGs. 10A-10B illustrate a non-limiting example of the mechanism of chicken embryo lethal orphan (CELO) virus genome replication. FIG. 10A shows a non-limiting schematic of the Ad5 genome, and its associated genetic elements, and a representation of a DNA replication polymerase involved in propagation of the Ad5 genome. FIG. 10B shows a non-limiting mechanism of adenovirus strand displacement replication.

[0034] FIGs. 11A-11H show non-limiting examples of vectors that may be used for production of rAAV particles. FIG. 11A shows an example of a vector comprising genetic elements used for pseudotyped avian rAAV particle production. FIGs. 11B, 11D, 11F, and 11F shows an example of an avian AAV packaging nucleic acid wherein the avian AAV rep gene has been cloned into the nucleic acid. FIGs. 11C, HE, and 11G shows an example of an avian rAAV genome including a heterologous nucleic acid comprising a transgene encoding green fluorescent protein (GFP).

[0035] FIGs. 12A-12D show fluorescent microscopy analyses of GFP expression delivered using a pseudotyped avian rAAV. FIG. 12A shows GFP expression in the chorioallantoic membrane (CAM) of an embryonated chicken egg that was inoculated with pseudotyped avian rAAV vectors. FIG. 12B shows GFP expression in chicken embryonic cells (CEC; derived from either brain or body tissues) transduced with pseudotyped avian rAAV particles comprising the indicated capsid proteins made in HEK293T cells. HEK293T cells comprising the corresponding pseudotyped avian rAAV vectors are shown as a control. FIG. 12C shows GFP expression in CECs transduced with pseudotyped avian rAAV particles. Avian QT6 cells comprising the corresponding pseudotyped avian rAAV vectors are shown as a control. FIG. 12D shows GFP expression in HEK293T cells contacted with a pseudotyped avian rAAV particle comprising a GFP gene flanked by avian AAV2 ITRs (left panel) and adult mouse brain cells contacted with a pseudotyped avian rAAV particle comprising a GFP gene and a mammalian AAV5 capsid protein (right panel).

[0036] FIGs. 13A-13D show a non-limiting example of a method for generating vectors for production of pseudotyped avian rAAV particles. FIG. 13A shows a representative cloning strategy for engineering a helper nucleic acid comprising pBeloBacl 1-CELO-gDNA-wt. FIG. 13B shows a map of the helper nucleic acid comprising pBeloBacl 1-CELO-gDNA-wt. FIG. 13C shows restriction enzyme digest and gel electrophoresis analysis confirming incorporation of CELO genomic DNA (gDNA) into the pBeloBacl 1 backbone. FIG. 13D shows GFP expression in CECs transduced with pseudotyped avian rAAV particles produced using helper functions of pHelper plasmid, CELO virus gDNA, or pBeloBacl 1-CELO-gDNA-wt.

[0037] DETAILED DESCRIPTION

[0038] The present disclosure provides engineered nucleic acids (e.g., transgenes and vectors) and compositions thereof that may be used to produce pseudotyped avian rAAV particles. Additionally, the present disclosure provides host cells (e.g., avian host cells) comprising said engineered nucleic acids and compositions, methods of producing pseudotyped avian rAAV particles, methods of treatment comprising administration of pseudotyped avian rAAV particles to a subject in need thereof, and kits related to the same. In some aspects, the disclosure relates to engineered nucleic acids for delivering rAAV production genes to a manufacturing system described herein (e.g., host cells, such as cells in avian eggs). Other aspects of the present disclosure provide manufacturing systems (e.g., host cells, such as isolated avian cells, and avian eggs) that may be used to produce pseudotyped avian rAAV particles using any of the engineered nucleic acids, compositions, and methods described herein. Accordingly, the present disclosure provides for gene therapies that are more compatible for in vivo applications than those produced through conventional means. Additionally, the present disclosure provides for cost-effective and scalable manufacturing methods for rAAV particle production (e.g., pseudotyped avian rAAV production) that provide high yields and purity levels which are adapted to large-scale manufacturing and / or are compatible with GLP / GMP guidelines.

[0039] For instance, in some aspects, the disclosure relates to pseudotyped avian rAAV particles and relates to compositions, manufacturing systems (e.g., an avian cell, such as an isolated avian cell or a cell in an avian egg), and / or methods involving one or more nucleic acids (e.g., one or more vectors, such as one or more plasmids) that comprise a heterologous nucleic acid flanked by avian AAV ITRs, an avian AAV rep protein gene sequence, and a mammalian AAV capsid protein gene sequence. In some embodiments, the one or more nucleic acids comprise one or more regulatory sequences, such as sequences that regulate expression and / or sequences that regulate replication including, but not limited to, replication that occurs in response to helper functions. For example, in some embodiments, the one or more sequences that regulate replication in response to helper functions include a CELO virus origin of replication. In some embodiments, the helper functions are provided by a helper virus, such as an adenovirus (e.g., an avian adenovirus, such as CELO helper virus). In some embodiments, the helper functions are provided by a helper nucleic acid.

[0040] Herein, general features of AAV and rAAV particles and genomes thereof are described. Then, a description of pseudotyped rAAV particles is provided. This is followed by an overview of pseudotyped avian rAAV particles which represents non-limiting aspects of the present invention and then a description of tools and methods for producing said pseudotyped avian rAAV particles.

[0041] Adeno-Associated Virus Particles

[0042] The term “AAV” is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all AAV subtypes including both naturally occurring and recombinant forms, unless otherwise indicated. An example of an AAV genome is found in FIG. 1A. Examples of AAVs include those that are found in an avian system (e.g., AAVs that can transfect an avian cell) (e.g., an avian AAV, which may be abbreviated as “AAAV”, such as those comprising an avian AAV capsid protein described herein), such as chicken cells, duck cells, goose cells, or quail cells. Further examples of AAVs include those that are found in a mammalian system (e.g., AAVs that can transfect a mammalian cell) (e.g., a mammalian AAV, such as those comprising a mammalian AAV capsid protein described herein), such as mouse cells, bovine cells, rat cells, canine cells, monkey cells, or human cells. The abbreviation “recombinant AAV (rAAV)” refers to recombinant adeno-associated virus which refers to AAV comprising a polynucleotide sequence not of AAV origin (e.g., a heterologous nucleic acid).

[0043] “rAAV genomes” comprise a heterologous nucleic acid flanked by 5' and 3' AAV inverted terminal repeats (ITRs). An example of an rAAV genome is provided in FIG. IB and FIG. 3A. In some embodiments, the 5' and 3' AAV ITRs may be alternatively referred to as “first” and “second” AAV ITRs, respectively (or, in the case of a pseudotyped avian rAAV genome or particle thereof, “first” and “second” avian AAV ITRs, respectively). In some embodiments, rAAV genomes may be linear or circular, single- stranded or double- stranded, and / or self-complementary. ITR sequences are about 145 bp in length. While the entire sequences encoding the ITRs are commonly used in engineering rAAVs, modification of these sequences is permissible and may be done using standard techniques (see, e.g., disclosures related to nucleic acid engineering and rAAV particle production in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Eisher et al., J Virol., 70:520 532 (1996)). Lor example, artificial ITRs may be engineered for tissue specificity. In some embodiments, a nucleic acid described herein (e.g., a nucleic acid comprised in a pseudotyped avian rAAV particle) comprises a heterologous nucleic acid flanked by a first AAV ITR and a second AAV ITR. In some embodiments, the first and second AAV ITRs are avian AAV ITRs.

[0044] The term “AAV particle” or “rAAV particle” refers to a viral particle comprising at least one AAV capsid protein and an encapsidated polynucleotide (e.g., an rAAV genome) which in some embodiments, may be administered to a subject and / or delivered to a selected target cell. Examples of AAV capsid proteins include VP1, VP2, and VP3 including, but not limited to, VP1, VP2, and VP3 proteins comprised in an avian AAV capsid and VP1, VP2, and VP3 proteins comprised in a mammalian AAV capsid. As used herein, “target cell” refers to cells that are contacted with rAAV particles (e.g., pseudotyped avian rAAV particles). Accordingly, target cells are the cells that receive the gene therapy provided in rAAV particles that were produced in host cells (e.g., such as those described herein) and include cells in culture and cells found in subjects. In some embodiments, a target cell (e.g., a cell contacted with a pseudotyped avian rAAV particle described herein) is a mammalian cell (e.g., a human cell). In some embodiments, a method described herein (e.g., a method of administering a pseudotyped avian rAAV particle to a subject, such as a mammalian subject) introduces a heterologous nucleic acid into one or more target cells in the subject. In some embodiments, the method is used to treat a disease or disorder described herein (e.g., by introducing a heterologous nucleic acid encoding a therapeutic RNA described herein). Functions of helper and packaging nucleic acids support AAV replication. Such functions include, without limitation, activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of capsid expression products, and AAV capsid assembly. Examples of suitable packaging nucleic acids are provided in Tables 1-5 and 7 and FIGs. 3B, 11B, 11D, 11F, and 11F.

[0045] A “helper vector” comprises a “helper nucleic acid” (e.g., a nucleic acid comprising an El gene, an E2A gene, an E4 gene, and / or a VA gene), which functions in trans for productive AAV replication and encapsidation. In some embodiments, a helper nucleic acid comprises a plurality of AAV helper genes described herein, such as two, three, four, five, six, seven, eight, nine, ten, or more than ten AAV helper genes. In some embodiments, a helper nucleic acid comprises one or more AAV helper genes (e.g., an El gene, an El gene, an E2A gene, an E4 gene, a VA gene, and / or an LTR gene) derived from an avian AAV helper virus (e.g., chicken embryo lethal orphan (CELO) virus). Preferably, the AAV helper nucleic acid supports efficient AAV vector production without generating any detectable wild-type AAV particles (e.g., AAV particles containing functional rep and capsid protein genes). Helper nucleic acids, and methods of making said nucleic acids, have been previously described and are commercially available (see, e.g., disclosures related to AAV helper nucleic acids and rAAV particle production in pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin- Binding Motif on Adeno- Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072- 11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid- Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno- Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini , Journal of Virology, Vol. 79, 5296-5303; Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adenoassociated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188).

[0046] The “packaging vector” comprises “a packaging nucleic acid” and provides nucleotide sequences (e.g., AAV rep and AAV capsid protein gene sequences) upon which an AAV is dependent for replication (e.g., accessory functions). In some embodiments, a packaging nucleic acid comprises an AAV rep gene sequence (e.g., an avian AAV rep gene sequence) and an AAV cap gene sequence (e.g., an avian AAV cap gene sequence or a mammalian AAV cap gene sequence). In some embodiments, a packaging nucleic acid comprises an avian AAV rep gene sequence and a mammalian AAV cap gene sequence. In some embodiments, an AAV rep gene sequence described herein comprises a sequence encoding a rep78 protein, a rep68 protein, a rep52 protein, and / or a rep40 protein. In some embodiments, an AAV cap gene sequence described herein encodes a VP1 protein, a VP2 protein, and / or a VP3 protein. In some embodiments, a packaging nucleic acid comprises an avian AAV rep protein gene sequence (e.g., avian AAV1 rep protein gene sequence or avian AAV2 rep protein gene sequence), and a mammalian AAV capsid protein gene sequence (e.g., mammalian AAV1 capsid protein gene sequence or mammalian AAV2 capsid protein gene sequence). In some embodiments, an avian rep protein gene sequence is a chicken AAV rep protein gene sequence, a duck AAV rep protein gene sequence, a goose AAV rep protein gene sequence, or quail AAV rep protein gene sequence. In some embodiments, a mammalian AAV capsid protein gene sequence is a mouse AAV capsid protein gene sequence, bovine AAV capsid protein gene sequence, monkey AAV capsid protein gene sequence, human AAV capsid protein gene sequence, or other mammalian AAV capsid protein gene sequence. In some embodiments, a packaging nucleic acid comprises one or more regulatory sequences described herein (e.g., a promoter, such as a major late promoter (MLP), and / or a bipartite leader (BPL) sequence).

[0047] In some embodiments, the components cultured in a “host cell” to package an rAAV genome in a capsid may be provided to the host cell in trans. In some embodiments, rAAV particles may be produced using the triple transfection method (see, e.g., disclosures related to rAAV particle production using triple transfection described in detail in U.S. Pat. No. 6,001,650) (see, e.g., FIG. 2A). Typically, the rAAV particles are produced by transfecting a host cell with an AAV vector (comprising a heterologous nucleic acid flanked by ITR elements) to be packaged into rAAV particles, and at least one AAV helper or packaging nucleic acid. Preferably two nucleic are used which include a helper nucleic acid and a packaging nucleic acid (see, e.g., FIG. 2A). Alternatively, in some embodiments, any one or more of the required components (e.g., heterologous nucleic acid flanked by AAV ITRs, rep sequences, cap sequences, and / or helper nucleic acids) may be provided by a stable host cell which has been engineered to contain one or more of the required components (FIG. 2B). Such a stable host cell will contain the required component(s) under the control of either an inducible promoter or a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory sequence suitable for use with a heterologous nucleic acid. Methods used to construct any engineered nucleic acid or rAAV particle thereof have also been previously described (see, e.g., disclosures related to nucleic acid engineering and rAAV particle production in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y). Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on this disclosure (see, e.g., disclosures related to rAAV particle production in K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745).

[0048] Recombinant Adeno- Associated Viruses Serotypes rAAV particles may be of a variety of serotypes. As used herein, the “serotype” of an rAAV refers to the type of capsid protein gene sequence used during rAAV particle production. Capsid protein gene sequences (e.g., those encoded by the packaging nucleic acid) encode the capsid proteins on the surface of the rAAV particle.

[0049] AAV serotypes have different “tropism” which refers to the ability of a specific rAAV particle capsid protein to bind to target cell receptors for viral transduction. As such, “pseudotyped rAAV particles” have been developed to generate rAAV particles with altered tropism, wherein the capsid protein is of a serotype heterologous to the serotype(s) of the ITRs. Previous work has demonstrated that pseudotyping other virus types also alters viral particle tropism in species, such as lentivirus (see, e.g., disclosures related to pseudotyped viruses in Takeuchi et al., (1997). Journal of Virology, 71(8): 6174-6178; Sandrin et al., (2002). Blood, 100(3): 823-832; Crawford et al., (2020). Viruses 12(513); and Li et al., (2018). Reviews in Medical Virology, 28(1): el963).

[0050] Examples of AAV serotypes include mammalian AAV1, mammalian AAV2, mammalian AAV3, mammalian AAV4, mammalian AAV5, mammalian AAV6, mammalian AAV7, mammalian AAV8, mammalian AAV9, mammalian AAV10, mammalian AAV11, mammalian AAV12, and mammalian AAV13. Non-limiting examples of rAAV pseudotypes include mammalian AAV2 / 1, mammalian AAV2 / 5, mammalian AAV2 / 6, mammalian AAV2 / 8, mammalian AAV2 / 9, mammalian AAV3 / 1, mammalian AAV3 / 5, mammalian AAV3 / 8, and mammalian AAV 3 / 9, wherein the slash denotes an rAAV genome of one serotype packaged in the capsid from a different serotype (e.g., an rAAV genome comprising AAV2 ITRs packaged in a capsid of AAV5 would be AAV2 / 5). Moreover, pseudotyped rAAV particles may be engineered with hybrid or mutant mammalian AAV capsid protein derivates, such as AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y- >F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV2(pentaYF), AAV2- BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F), AAV6(T492V+S663V), AAV6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8-C&G(T494V), AAV8-M3, AAV8(Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B, and AAVr3.45. Such AAV serotypes and derivatives / pseudotypes as well as methods for producing them have been previously described (see, e.g., disclosures related to engineered nucleic acids for rAAV particle production in Mol. Ther. 2012 Apr;20(4):699- 708. doi:

[0051] 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ; Duan et al, J. Virol., 75:7662-7671, 2001; Halbert et al, J. Virol., 74:1524-1532, 2000; Zolotukhin et al, Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001; see, e.g., US Patent Publication No.: US 2005 / 0100890 Al; International Publication No.: WO 01 / 83692 A2; US Patent Publication No.: US 2003 / 0103939 Al; and Miller (1996). Proc. Natl. Acad. Sci., 93: 11407-11413).

[0052] In some embodiments, an rAAV particle described herein comprises a capsid protein of a mammalian AAV serotype. In some embodiments, the mammalian AAV serotype is mammalian AAV1. In some embodiments, the mammalian AAV serotype is mammalian AAV2. In some embodiments, the mammalian AAV serotype is mammalian AAV3. In some embodiments, the mammalian AAV serotype is mammalian AAV4. In some embodiments, the mammalian AAV serotype is mammalian AAV5. In some embodiments, the mammalian AAV serotype is mammalian AAV6. In some embodiments, the mammalian AAV serotype is mammalian AAV7. In some embodiments, the mammalian AAV serotype is mammalian AAV8. In some embodiments, the mammalian AAV serotype is mammalian AAV9. In some embodiments, the mammalian AAV serotype is mammalian AAV 10. In some embodiments, the mammalian AAV serotype is mammalian AAV11. In some embodiments, the mammalian AAV serotype is mammalian AAV12. In some embodiments, the mammalian AAV serotype is mammalian AAV13. In some embodiments, an rAAV particle described herein that comprises a capsid protein of a mammalian AAV serotype further comprises a heterologous nucleic acid described herein, wherein the heterologous nucleic acid is flanked by avian AAV ITRs.

[0053] In some embodiments, an rAAV particle described herein comprises a hybrid or mutant capsid protein derivative of a mammalian AAV serotype. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVrh.10 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVrh.74 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVhu.14 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV3a / 3b capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVrh32.33 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV-HSC15 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV- HSC17 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVhu.37 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVrh.8 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is CHt-P6, AAV2.5 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV6.2 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV2i8 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV-HSC15 / 17 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVM41 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV9.45 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV6(Y445F / Y731F) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV2.5T capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV-HAE1 / 2 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV clone 32 / 83 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVShHIO capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV2 (Y->F) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV8 (Y733F) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV2.15 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV2.4 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVM41 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV2(pentaYF) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV2- BCDG(T491V+K556R) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV5-M2 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV5(Y719F) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV6(T492V+S663V) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV6(T492V+Y705F+Y731F) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV6(S551V+S663V) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV8-C&G(T494V) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV8-M3 capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV8(Y733F) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV8(T494V+Y733F) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV8(Y275F+Y447F+Y733F) capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAV9-PHP.B capsid protein. In some embodiments, the hybrid or mutant capsid protein derivative of a mammalian AAV serotype is an AAVr3.45 capsid protein. In some embodiments, an rAAV particle described herein that comprises a hybrid or mutant capsid protein derivative of a mammalian AAV serotype further comprises a heterologous nucleic acid described herein, wherein the heterologous nucleic acid is flanked by avian AAV ITRs. In some embodiments, an rAAV particle described herein comprises ITRs of an avian AAV serotype. In some embodiments, the avian AAV serotype is avian AAV1. In some embodiments, the avian AAV serotype is avian AAV2. In some embodiments, the avian AAV serotype is avian AAV3. In some embodiments, the avian AAV serotype is avian AAV4. In some embodiments, the avian AAV serotype is avian AAV5. In some embodiments, the avian AAV serotype is avian AAV6. In some embodiments, the avian AAV serotype is avian AAV7. In some embodiments, the avian AAV serotype is avian AAV8. In some embodiments, the avian AAV serotype is avian AAV9. In some embodiments, the avian AAV serotype is avian AAV10.

[0054] Pseudotyped Avian Recombinant Adeno- Associated Virus Particles

[0055] Avian AAVs have been found in various bird species. Avian AAVs associated with avian adenoviruses (Aviadenoviridae) infect chickens, ducks, quail, and other avian species. Said AAVs are designated herein as “avian AAV” and may be abbreviated as “AAAV”.

[0056] The present disclosure provides for “pseudotyped avian rAAV particles” which refer to particles comprising avian AAV ITRs flanking a heterologous nucleic acid, and a non-avian AAV capsid protein (e.g., a mammalian AAV capsid protein). An example of a pseudotyped avian rAAV genome is provided in FIGs. IB, FIG. 3A, and Table 6, and FIGs. 11C, 1 IE, and 11G. In some embodiments, methods described herein involve production of pseudotyped avian rAAV particles using a packaging nucleic acid comprising a sequence encoding avian AAV rep protein (see, e.g., the nucleic acid sequence set forth in SEQ ID NO: 9).

[0057] As used herein, an “avian AAV ITR” refers to any 5' or 3' AAV ITR found in an avian AAV, such as AAVs that infect chickens, quails, ducks, and geese. Non-limiting examples of avian AAV ITR sequence include the nucleic acid sequences set forth in SEQ ID NOs: 24 and 30. Non-limiting examples of a 5’ or “first” avian AAV ITR sequence include the nucleic acid sequence set forth in SEQ ID NO: 24. Non-limiting examples of a 3’ or “second” avian AAV ITR sequence include the nucleic acid sequence set forth in SEQ ID NO: 30. In some embodiments, a pseudotyped avian rAAV particle described herein comprises avian AAV1 ITRs, avian AAV2 ITRs, avian AAV3 ITRs, avian AAV4 ITRs, avian AAV5 ITRs, avian AAV6 ITRs, avian AAV7 ITRs, avian AAV8 ITRs, avian AAV9 ITRs, or avian AAV10 ITRs. In some embodiments, a pseudotyped avian rAAV particle described herein comprises a mammalian AAV1 capsid protein, a mammalian AAV2 capsid protein, a mammalian AAV3 capsid protein, a mammalian AAV4 capsid protein, a mammalian AAV5 capsid protein, a mammalian AAV6 capsid protein, a mammalian AAV7 capsid protein, a mammalian AAV8 capsid protein, a mammalian AAV9 capsid protein, a mammalian AAV10 capsid protein, a mammalian AAV 11 capsid protein, a mammalian AAV 12 capsid protein, a mammalian

[0058] AAV13 capsid protein, a AAVrh.10 capsid protein, an AAVrh.74 capsid protein, an AAVhu.14 capsid protein, an AAV3a / 3b capsid protein, an AAVrh32.33 capsid protein, an AAV-HSC15 capsid protein, an AAV- HSC17 capsid protein, an AAVhu.37 capsid protein, an AAVrh.8 capsid protein, an CHt-P6 capsid protein, an AAV2.5 capsid protein, an AAV6.2 capsid protein, an AAV2i8 capsid protein, an AAV-HSC15 / 17 capsid protein, an AAVM41 capsid protein, an AAV9.45 capsid protein, an AAV6(Y445F / Y731F) capsid protein, an AAV2.5T capsid protein, an AAV-HAE1 / 2 capsid protein, an AAV clone 32 / 83 capsid protein, an AAVShHIO capsid protein, an AAV2 (Y->F) capsid protein, an AAV8 (Y733F) capsid protein, an AAV2.15 capsid protein, an AAV2.4 capsid protein, an AAVM41 capsid protein, an AAV2(pentaYF) capsid protein, an AAV2-BCDG(T491V+K556R) capsid protein, an AAV5-M2 capsid protein, an AAV5(Y719F) capsid protein, an AAV6(T492V+S663V) capsid protein, an AAV6(T492V+Y705F+Y731F) capsid protein, an AAV6(S551V+S663V) capsid protein, an AAV8-C&G(T494V) capsid protein, an AAV8-M3 capsid protein, an AAV8(Y733F) capsid protein, an AAV8(T494V+Y733F) capsid protein, an AAV8(Y275F+Y447F+Y733F) capsid protein, an AAV9-PHP.B capsid protein, or an AAVr3.45 capsid protein.

[0059] In some embodiments, pseudotyped avian rAAV particles of the present disclosure have several advantages over previous rAAV particles. In some embodiments, pseudotyped avian rAAV particles exhibit increased host-range and transduction efficiency relative to conventional rAAV particles. In some embodiments, pseudotyped avian rAAV particles exhibit altered tropism that is not achievable with conventional rAAV particles. In some embodiments, pseudotyped avian rAAV particles exhibit lower cytotoxicity as compared to conventional rAAV gene therapies. In some embodiments, pseudotyped avian rAAV particles are better at evading inactivation by non-specific complement mechanisms in serum than conventional rAAV gene therapies. In other embodiments, pseudotyped avian rAAV particles are used to enable safer study of pathogens by transferring a viral gene from a pathogen into the genome of a pseudotyped avian rAAV.

[0060] In some embodiments, a nucleic acid encoding one or more elements that are useful to make rAAV particles in avian cells or eggs (e.g., a vector comprising an rAAV genome, a rep gene, a cap gene, and / or one or more helper genes) can also include non- AAV ITR sequences (from another virus) that are responsive to the addition of one or more helper functions. In some embodiments, the non-AAV ITRs are avian virus ITRs. In some embodiments, the non- AAV ITRs are avian virus ITRs are from Fowl Adenovirus 1 (FAdV), also known as chicken embryo lethal orphan (CELO) virus. Non-limiting examples of CELO virus ITRs include the nucleic acid sequences set forth in SEQ ID NOs: 4-5. In some embodiments, the non-AAV ITRs promote replication of the plasmid they are located on in response to the presence of one or more helper functions (e.g., encoded by one or more non- AAV helper genes, for example from the same virus as the non-AAV ITRs).

[0061] Engineered Nucleic Acids

[0062] Aspects of the present disclosure relate to engineered nucleic acids that can be used to produce pseudotyped avian rAAV particles. As used herein, an “engineered nucleic acid” sequence may encompass a DNA or RNA sequence. As used herein, the term “engineered” means artificially produced. As such, with respect to nucleic acids, the term “engineered” may also mean: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; or (iii) synthesized by, for example, chemical synthesis. An engineered nucleic acid is one which has been manipulated by recombinant DNA techniques well known in the art e.g., by genetic engineering techniques using restriction enzymes, ligases, and similar recombinant techniques as described by, for example, disclosures related to nucleic acid engineering in Sambrook et al., Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; (1989), or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989), and DNA Cloning: A Practical Approach, Volumes I and II (ed. D. N. Glover) IREL Press, Oxford, (1985); each of which is incorporated herein by reference. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known, or for which polymerase chain reaction (PCR) primer sequences may be designed, is considered engineered. However, a nucleic acid sequence existing in its native state in its natural host is not an engineered nucleic acid. An engineered nucleic acid may be substantially purified but need not be. Accordingly, nucleic acids may be considered engineered when a non-naturally occurring gene sequence has been generated in a laboratory setting. Such non-naturally occurring gene sequences comprise, in general, select components derived from one or more sources of genetic information to yield a nucleic acid encoding a protein with unique functions not found in nature. In some embodiments, engineered nucleic acids may be codon-optimized. In some embodiments, a codon-optimized sequence in an engineered nucleic acid is codon- optimized based on the codon usage bias of a manufacturing system described herein. In some embodiments, a codon-optimized sequence in an engineered nucleic acid is codon-optimized based on the codon usage bias of a target cell described herein (e.g., a target cell in a subject administered an rAAV particle described herein). Non-limiting examples of engineered nucleic acids include transgenes, vectors (e.g., vectors comprising heterologous nucleic acids or transgenes thereof), and recombinant viral genomes. In some embodiments, engineered nucleic acids of the present disclosure comprise approximately 1-10,000 nucleotides. In some embodiments, engineered nucleic acids comprise approximately 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1,000, 1,000-1,100,

[0063] 1.100-1,200, 1,200-1,300, 1,300-1,400, 1,400-1,500, 1,500-1,600, 1,600-1,700, 1,700-1,800,

[0064] 1.800-1,900, 1,900-2,000, 2,000-2,100, 2,100-2,200, 2,200-2,300, 2,300-2,400, 2,400-2,500,

[0065] 2.500-2,600, 2,600-2,700, 2,700-2,800, 2,800-2,900, 2,900-3,000, 3,000-3,100, 3,100-3,200,

[0066] 3.200-3,300, 3,300-3,400, 3,400-3,500, 3,500-3,600, 3,600-3,700, 3,700-3,800, 3,900-4,000, 4,000-4,100, 4,100-4,200, 4,200-4,300, 4,300-4,400, 4,400-4,500, 4,500-4,600, 4,600-4,700, 4,700-4,800, 4,800-4,900, 4,900-5,000, 5,000-5,100, 5,100-5,200, 5,200-5,300, 5,300-5,400, 5,400-5,500, 5,500-5,600, 5,600-5,700, 5,700-5,800, 5,800-5,900, 5,900-6,000, 6,000-6,100,

[0067] 6.100-6,300, 6,200-6,300, 6,300-6,400, 6,400-6,500, 6,500-6,600, 6,600-6,700, 6,700-6,800,

[0068] 6.800-6,900, 6,900-7,000, 7,000-7,100, 7,100-7,200, 7,200-7,300, 7,300-7,400, 7,400-7,500,

[0069] 7.500-7,600, 7,600-7,700, 7,700-7,800, 7,800-7,900, 7,900-8,000, 8,000-8,100, 8,100-8,200,

[0070] 8.200-8,300, 8,300-8,400, 8,400-8,500, 8,500-8,600, 8,600-8,700, 8,700-8,800, 8,800-8,900, 8,900-9,000, 9,000-9,100, 9,100-9,200, 9,200-9,300, 9,300-9,400, 9,400-9,500, 9,500-9,600, 9,600-9,700, 9,700-9,800, 9,800-9,900, or 9,900-10,000 nucleotides. However, in some embodiments, larger heterologous nucleic acids could be used, for example, 10-20 kb or larger.

[0071] In some embodiments, an engineered nucleic acid sequence comprises DNA. In some embodiments, an engineered nucleic acid sequence comprises RNA. In some embodiments, an engineered nucleic acid sequence comprises a combination of DNA and RNA. In some embodiments, an engineered nucleic acid comprises double- stranded DNA. In some embodiments, an engineered nucleic acid comprises single-stranded DNA. In some embodiments, an engineered nucleic acid comprises double- stranded RNA. In some embodiments, an engineered nucleic acid comprises single-stranded RNA. In some embodiments, an engineered nucleic acid comprises a double-stranded nucleic acid comprising a combination of DNA and RNA nucleotides. In some embodiments, an engineered nucleic acid comprises a double- stranded nucleic acid (e.g., DNA, RNA, or a combination of DNA / RNA) comprising gaps (e.g., stretches of sequence about, 1, 5, 10, 25, 50, 100 or more nucleotides in length) of single- stranded sequence at any position within the molecule. In some embodiments, an engineered nucleic acid is linear. In some embodiments, an engineered nucleic acid is circular. In some embodiments, an engineered nucleic acid comprises at least one singlestranded nick. In some embodiments, to produce a pseudotyped avian rAAV particle, a “plurality” of engineered nucleic acids are contacted with a suitable manufacturing system (e.g., a host cell or avian egg) described herein. The plurality of engineered nucleic acids comprises at least two engineered nucleic acids referred to as a “first engineered nucleic acid” and a “second engineered nucleic acid.” In some embodiments, the plurality of engineered nucleic acids (e.g., the first and second engineered nucleic acids) is provided on separate nucleic acids (e.g., two different vectors, such as wherein one vector comprises the first nucleic acid and the other vector comprises the second nucleic acid). In some embodiments, the first engineered nucleic acid comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NO: 23. In some embodiments, the first engineered nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the second engineered nucleic acid comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 1, 15, 17, 19, 23, or 32. In some embodiments, the second engineered nucleic acid comprises the nucleic acid sequence of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1, 15, 17, 19, 23, or 32. In some embodiments, the first engineered nucleic acid further comprises a 5’ CELO virus ITR and a 3’ CELO virus ITR. In some embodiments, the second engineered nucleic acid further comprises a 5’ CELO virus ITR and a 3’ CELO virus ITR.

[0072] First Engineered Nucleic Acid

[0073] In some embodiments, the “first engineered nucleic acid” of the plurality comprises a heterologous nucleic acid described herein (e.g., see FIG. IB and Fig. 3A). In some embodiments, the first engineered nucleic acid comprises a heterologous nucleic acid flanked by first and second avian AAV ITRs (e.g., naturally occurring or modified avian AAV ITRs). In some embodiments the first engineered nucleic acid comprises a heterologous nucleic acid flanked by avian AAV1 ITRs, avian AAV2 ITRs, avian AAV3 ITRs, avian AAV4 ITRs, avian AAV5 ITRs, avian AAV6 ITRs, avian AAV7 ITRs, avian AAV8 ITRs, avian AAV9 ITRs, or avian AAV10 ITRs. In some embodiments, the first engineered nucleic acid comprises a first and second artificial avian AAV ITR that is engineered for tissue-specificity. In some embodiments, the first engineered nucleic acid comprises a first and second artificial avian AAV ITR that is engineered for production of a self-complementary rAAV genome. In some embodiments, the first avian AAV ITR comprises the nucleic acid sequence of SEQ ID NO: 24. In some embodiments, the second avian AAV ITR comprises the nucleic acid sequence of SEQ

[0074] ID NO: 30.

[0075] Heterologous Nucleic Acids

[0076] A “heterologous nucleic acid” refers to any DNA sequence that is not normally found between flanking AAV ITRs (e.g., avian AAV ITRs). In some embodiments, a heterologous nucleic acid is an engineered nucleic acid sequence. In some embodiments, a heterologous nucleic acid is a naturally occurring nucleic acid sequence (e.g., a non-engineered sequence) that is not normally found between AAV ITRs (e.g., avian AAV ITRs). In some embodiments, a heterologous nucleic acid may contain segments of DNA taken from different organisms. In some embodiments, a heterologous nucleic acid may comprise any combination of naturally- occurring and engineered DNA sequences.

[0077] In some embodiments, heterologous nucleic acids of the present disclosure comprise 1- 5,000 nucleotides. In some embodiments, heterologous nucleic acids comprise approximately 1- 10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1,000, 1,000-1,100, 1,100-1,200, 1,200- 1,300, 1,300-1,400, 1,400-1,500, 1,500-1,600, 1,600-1,700, 1,700-1,800, 1,800-1,900, 1,900- 2,000, 2,000-2,100, 2,100-2,200, 2,200-2,300, 2,300-2,400, 2,400-2,500, 2,500-2,600, 2,600- 2,700, 2,700-2,800, 2,800-2,900, 2,900-3,000, 3,000-3,100, 3,100-3,200, 3,200-3,300, 3,300- 3,400, 3,400-3,500, 3,500-3,600, 3,600-3,700, 3,700-3,800, 3,900-4,000, 4,000-4,100, 4,100- 4,200, 4,200-4,300, 4,300-4,400, 4,400-4,500, 4,500-4,600, 4,600-4,700, 4,700-4,800, 4,800- 4,900, or 4,900-5,000 nucleotides. However, in some embodiments, larger heterologous nucleic acids could be used, for example, 5-10 kb or larger.

[0078] In some embodiments, a heterologous nucleic acid comprises at least one transgene. As used herein, “transgene” refers to a DNA sequence which encodes an RNA to be expressed in a cell. In some embodiments, a heterologous nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more transgenes. Examples of sequences found in transgenes include sequences encoding interfering RNAs (e.g., shRNAs, siRNAs, miRNAs, ncRNAs, piRNAs, pro-siRNAs, etc.), exonskipping RNAs, enzymatic RNAs, guide RNAs or gRNAs (e.g., sgRNAs) of a CRISPR / Cas editing system (e.g., an editing system comprising a Cas protein and a gRNA including, but not limited to, a Cas9-based genome editing system and derivatives thereof or a Cas 12-based genome editing system and derivatives thereof, such as Cas9- or Cas 12-based editing systems for base editing and prime editing including, but not limited to, editing systems that utilize a StlCas9), small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and messenger RNAs (mRNAs). In some embodiments, a transgene comprises a cDNA sequence. In some embodiments, RNA expressed from a transgene is not translated into a protein. In some embodiments, a transgene encodes an mRNA that is translated into a protein. In some embodiments, a transgene may comprise multiple DNA sequences (e.g., a transgene that encodes multiple RNA species). In some embodiments, heterologous nucleic acids comprise a plurality of sequences wherein at least one sequence (e.g., a transgene) of the plurality are expressed in a cell and at least one other sequence of the plurality are not expressed in the cell (e.g., repetitive elements, repair templates, etc.).

[0079] In some embodiments, a transgene encodes a selectable marker. In some embodiments, a selectable marker is used to assay heterologous nucleic acid expression in a cell or subject and / or sort transduced host and / or target cells. Examples of selectable markers include cell surface proteins (e.g., an antibody or antigen-binding fragment thereof, receptors, membrane proteins which become glycosylated upon expression in a cell, etc.) and fluorescent markers (e.g., mNeonGreen, GFP (e.g., SEQ ID NO: 28), EGFP, Superfold GFP, Azami Green, mWasabi, TagGFP, TurboGFP, acGFP, zsGreen, T-sapphire, EBFP, EBFP2, Azurite, TagBFP, ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyanl, TagCFP, mTFPl, EYFP, mCitrine, TagYFP, phiYFP, zsYellowl, mBanana, Kusabira Orange, mOrange, dTomato, DsRed, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFPl, mCherry, HcRedl, iRFP720, smURFP, and AQ143).

[0080] In some embodiments, a transgene encodes a therapeutic RNA and / or protein. As used herein, a “therapeutic RNA” or “therapeutic protein” leads to a physiological change that is associated with or expected to at least partially, if not fully, remedy at least one symptom associated with a disease, disorder, or condition. A therapeutic RNA refers to an RNA expressed from a transgene that is therapeutic as an RNA upon expression in a target cell and without being translated into a protein. A therapeutic protein refers to any proteinaceous molecule that is translated from an RNA expressed from a transgene which is therapeutic upon translation in a target cell. A therapeutic RNA or protein may be therapeutic for any disease, disease, or condition described herein upon administration to a subject in need thereof (e.g., a mammalian subject, such as a human subject).

[0081] Non-limiting examples of therapeutic RNAs include any antisense RNA (e.g., shRNA, miRNA, etc.), exon-skipping RNA, enzymatic RNA, gRNA, snRNA, rRNA, tRNA, and mRNA. In some embodiments, a therapeutic RNA modulates the expression of a gene implicated in a disease, disorder, or condition described herein. Non-limiting examples of therapeutic proteins include enzymes (e.g., proteases, signaling proteins, transcriptional regulators, Cas9 or derivatives thereof, Casl2 or derivates thereof, base editors, prime editors, etc.), enzymatic domains, enzyme substrates, hormones, receptors (e.g., chimeric antigen receptors), components of gene editing ribonucleoprotein complexes (e.g., Cas9 or derivatives thereof, Casl2 or derivatives thereof, base editors, prime editors, etc), peptibodies, growth factors, clotting factors, cytokines, chemokines, activating or inhibitory peptides acting on cell surface receptors or ion channels, cell-permeable peptides targeting intracellular processes, thrombolytics, bone morphogenetic proteins, Fc-fusion proteins, anticoagulants, and antibodies or antigen-binding fragments thereof. In some embodiments, a therapeutic RNA or protein is selected for the purposes of gene replacement therapy. In some embodiments, the therapeutic protein is selected for the purposes of vaccine production against a human pathogen (e.g., a protein, or fragment thereof, comprising an antigen of a human pathogen). In some embodiments, the therapeutic RNA or protein is selected for anti-cancer therapy.

[0082] However, in some embodiments, a heterologous nucleic acid is not transcribed into an RNA and / or does not promote synthesis of a corresponding protein. In some embodiments, a heterologous nucleic acid comprises at least one sequence that is not expressed in a cell. In some embodiments, a heterologous nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sequences not expressed in a cell.

[0083] In some embodiments, a heterologous nucleic acid comprises a regulatory sequence described herein. In some embodiments, a heterologous nucleic acid comprises one or more regulatory sequences that regulate transcription (e.g., any one or more of a promoter, an enhancer, a transcription factor binding sequence, a transcriptional start sequence, transcription termination sequence, etc.), one or more regulatory sequences that regulate translation (e.g., any one or more of a 5’ UTR, a translation initiation regulatory sequence, a Kozack sequence, a start codon, a ribosome binding site, a 3’ UTR, a translation termination sequence, a stop codon, etc.), and / or one or more regulatory sequences that regulate splicing (e.g., any one or more of a binding site for small nuclear ribonucleoproteins, splicing acceptor sites, splicing donor sites, etc.) of an RNA encoded by a sequence in a heterologous nucleic acid. In some embodiments, a promoter comprised in a heterologous nucleic acid is one that is active in a target cell described herein (e.g., a target cell in a subject administered a pseudotyped avian rAAV particle administered to a mammalian subject, such as a human subject). In some embodiments, a heterologous nucleic acid comprises a constitutive promoter (e.g., a Herpes Simplex virus (HSV) promoter, a thymidine kinase (TK) promoter, a Simian Virus 40 (SV40) promoter, a

[0084] Mouse Mammary Tumor Virus (MMTV) promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), a dihydrofolate reductase promoter, a P-actin promoter, a phosphoglycerate kinase (PGK) promoter, a CAG promoter, a human elongation factor- 1 alpha (EFla) promoter, an RNA pol II promoter, an RNA pol III promoter, a U6 promoter, a Hl promoter, an RNA pol II promoter, a chicken P-actin (CBA) promoter, etc.), an inducible promoter (e.g., cytochrome P450 gene promoters, heat shock protein gene promoters, metallothionein gene promoters, hormone-inducible gene promoters, a zinc-inducible sheep metallothionine (MT) promoter, a dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, a T7 polymerase promoter system, a ecdysone insect promoter, a tetracycline-inducible promoter, a rapamycin-inducible promoter, etc.), or a tissue-specific promoter (e.g., a neuron- specific promoter, a skeletal muscle- specific promoter, a smooth muscle- specific promoter, an immune cell-specific promoter, a cardiac muscle-specific promoter, etc.). In some embodiments, a tissue-specific promoter in a heterologous nucleic acid is a promoter that is active in a target cell that a pseudotyped avian rAAV particle (comprising the heterologous nucleic acid) binds to. In some embodiments, a heterologous nucleic acid comprises a regulatory sequence operably linked to a gene sequence encoding an RNA. In some embodiments, a heterologous nucleic acid comprises a regulatory sequence which is not operably linked to a gene sequence encoding an RNA. In some embodiments, an engineered nucleic acid comprises at least one splice donor and / or splice acceptor site. In some embodiments, a regulatory sequence comprises at least one of a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, a gene sequence encoding an RNA, and a Proudfoot poly(A) signal. In some embodiments, a heterologous nucleic acid comprises at least one of the nucleic acid sequences set forth in SEQ ID NOs: 25-29. In some embodiments, a heterologous nucleic acid comprises a nucleic acid used as a substrate for genetic engineering (e.g., a repair template used for homology-directed repair mechanisms).

[0085] In some embodiments, a transgene comprises one or more gene sequences (e.g., those coding for interfering RNAs, mRNAs, etc.) operably linked to at least one regulatory sequence described herein. In some embodiments, a transgene comprises at least one regulatory sequence located at the 5' end such that it is located upstream of a gene sequence (e.g., in the 5' untranslated region (UTR)). In some embodiments, a transgene comprises at least one regulatory sequence located at the 3' end such that it is located downstream of the gene sequence (e.g., in the 3' UTR). In some embodiments, a transgene comprises at least one regulatory at the 5' end and at least one regulatory sequence located at the 3' end such that regulatory sequences are located upstream and downstream of the gene sequence. In some embodiments, a regulatory sequence (e.g., an intron) at the 5' end of the transgene comprises a miRNA. In some embodiments, a regulatory sequence at the 3' end of the transgene comprises a miRNA or a safety kill switch. In some embodiments, a transgene comprises from 5' to 3', in relative order, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, a gene sequence encoding an RNA, and a Proudfoot poly(A) signal. In some embodiments, a transgene nucleic acid comprises at least one of the nucleic sequences set forth in SEQ ID NOs: 25-29. In some embodiments, a transgene comprises at least one splice donor and / or splice acceptor site.

[0086] Second Engineered Nucleic Acid

[0087] In some embodiments, the “second engineered nucleic acid” of the plurality comprises the avian AAV rep and / or AAV capsid protein gene sequences. In some embodiments, the second engineered nucleic acid is a packaging nucleic acid. In some embodiments, a gene sequence encoding the avian AAV rep protein is encoded by the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, a gene sequence encoding a capsid protein may correspond to any AAV capsid protein described herein. In some embodiments, a gene sequence encodes VP1, VP2, and / or VP3. In some embodiments, a gene sequence encodes one or more mammalian AAV capsid proteins. In some embodiments, a gene sequence encodes at least one AAV capsid protein selected from the group consisting of mammalian AAV1 (e.g., SEQ ID NO: 32), mammalian AAV5, mammalian AAV6 (e.g., SEQ ID NO: 10), mammalian AAV7 (e.g., SEQ ID NO: 16), mammalian AAV9 (e.g., SEQ ID NO: 20), and mammalian AAV8-M3 (e.g., SEQ ID NO: 18). In some embodiments, the second engineered nucleic acid may comprise an avian AAV rep gene and a non-avian AAV cap gene, such as a mammalian AAV2 cap gene, a mammalian AAV3 cap gene, a mammalian AAV4 cap gene, a mammalian AAV4 cap gene, a mammalian AAV5 cap gene, a mammalian AAV6 cap gene, a mammalian AAV7 cap gene, a mammalian AAV8 cap gene, a mammalian AAV 10 cap gene, a mammalian AAV 11 cap gene, a mammalian AAV 12 cap gene, a mammalian AAV 13 cap gene, or a gene encoding a hybrid or mutant mammalian AAV cap derivate, such as an AAVrh.10 cap gene, an AAVrh.74 cap gene, an AAVhu.14 cap gene, an AAV3a / 3b cap gene, an AAVrh32.33 cap gene, an AAV-HSC15 cap gene, an AAV- HSC17 cap gene, an AAVhu.37 cap gene, an AAVrh.8 cap gene, CHt-P6 cap gene, an AAV2.5 cap gene, an AAV6.2 cap gene, an AAV2i8 cap gene, an AAV-HSC15 / 17 cap gene, an AAVM41 cap gene, an AAV9.45 cap gene, an AAV6(Y445F / Y731F) cap gene, an AAV2.5T cap gene, an AAV-HAE1 / 2 cap gene, an AAV clone 32 / 83 cap gene, an AAVShHIO cap gene, an AAV2 (Y->F) cap gene, an AAV8 (Y733F) cap gene, an AAV2.15 cap gene, an AAV2.4 cap gene, an AAVM41 cap gene, an AAV2(pentaYF) cap gene, an AAV2- BCDG(T491V+K556R) cap gene, an AAV5-M2 cap gene, an AAV5(Y719F) cap gene, an AAV6(T492V+S663V) cap gene, an AAV6(T492V+Y705F+Y731F) cap gene, an AAV6(S551V+S663V) cap gene, an AAV8-C&G(T494V) cap gene, an AAV8(Y733F) cap gene, an AAV8(T494V+Y733F) cap gene, an AAV8(Y275F+Y447F+Y733F) cap gene, an AAV9-PHP.B cap gene, or an AAVr3.45 cap gene. In some embodiments, the AAV capsid protein gene sequence is encoded on the same strand as one or more AAV rep gene sequences. In some embodiments, the AAV capsid protein gene sequence and one or more AAV rep gene sequences are encoded on different strands of a single nucleic acid.

[0088] In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein comprises one or more AAV rep genes and / or one or more AAV cap genes which are operably linked to one or more regulatory sequences described herein. In some embodiments, the one or more regulatory sequences comprise regulatory sequences that regulate transcription (e.g., any one or more of a promoter, an enhancer, a transcription factor binding sequence, a transcriptional start sequence, transcription termination sequence, etc.), regulatory sequences that regulate translation (e.g., any one or more of a 5’ UTR, a translation initiation regulatory sequence, a Kozack sequence, a start codon, a ribosome binding site, a 3’ UTR, a translation termination sequence, a stop codon, etc.), and / or one or more regulatory sequences that regulate splicing (e.g., any one or more of a binding site for small nuclear ribonucleoproteins, splicing acceptor sites, splicing donor sites, an intron etc.) of an RNA encoded by the one or more AAV rep gene sequences and / or the one or more AAV cap gene sequences. In some embodiments, the one or more regulatory sequences comprise a promoter, such as a constitutive promoter, a tissue- specific promoter, or an inducible promoter described herein. In some embodiments, the promoter is a mouse mammary tumor virus (MMTV) promoter. In some embodiments, the promoter is a major late promoter (MLP). In some embodiments, the one or more regulatory sequences comprise an intron (e.g., an intron flanked by a splicing acceptor site and a splicing donor site). In some embodiments, the intron is a human collagen gene intron. In some embodiments, the intron is a beta globin gene intron. In some embodiments, the intron is a lambda intron.

[0089] Helper Nucleic Acids

[0090] In some embodiments, a helper nucleic acid comprising at least one helper gene sequence is also contacted with a manufacturing system to produce pseudotyped avian rAAV particles. Examples of helper nucleic acids described herein, as well compositions thereof and methods of utilizing the helper nucleic acids, for production of a pseudotyped avian rAAV particle are illustrated in FIGs. 2A-2C. These examples should not be considered limiting as, in some embodiments, one or more of the components of the nucleic acids illustrated in FIGs. 2A- 2C are omitted or replaced with one or more different helper nucleic acid components. For example, in some embodiments, FIG. 2A illustrates a packaging cell lacking an El gene from adenovirus 5 (Ad5). However, in some embodiments, a packaging cell comprises an El gene (e.g., an El gene from adenovirus 5 (Ad5)). In some embodiments, a packaging cell lacks one or more helper genes described herein other than an El gene (e.g., an E2A gene, an E4 gene, a VA gene, and / or an LTR gene). As a further example, in some embodiments, a helper nucleic acid (as illustrated in FIG. 2B or FIG. 2C) lacks an El gene, an E2A gene, or a VA gene.

[0091] In some embodiments, a helper nucleic acid comprises a plurality of AAV helper genes described herein, such as two, three, four, five, six, seven, eight, nine, ten, or more than ten AAV helper genes. In some embodiments, helper gene sequences comprise at least one AAV helper gene, such as an El gene, an E2A gene, an E4 gene, a VA gene, an LTR gene, or any combination thereof. In some embodiments, a helper nucleic acid is an adenovirus helper nucleic acid, such as an avian adenovirus helper nucleic acid. In some embodiments, a helper nucleic acid comprises one or more AAV helper genes (e.g., an El gene, an El gene, an E2A gene, an E4 gene, a VA gene, and / or an LTR gene) derived from an avian AAV helper virus (e.g., chicken embryo lethal orphan (CELO) virus). In some embodiments, the avian adenovirus helper nucleic acid is a CELO helper virus nucleic acid. In some embodiments, the helper virus nucleic acid is provided on a vector. In some embodiments, the helper nucleic acid comprises a sequence with least 75% identity to a nucleic acid sequence set forth in SEQ ID NO: 36 (e.g., at least 75% identity to a helper sequence comprised in the nucleotide sequence set forth in SEQ ID NO: 36, such as a helper sequence comprising at least one of El, E2A, E4, or VA genes).

[0092] In some embodiments, a plurality of helper gene sequences is provided on a helper nucleic acid or a “third engineered nucleic acid”. In some embodiments, a helper nucleic acid may be separate (e.g., on a different vector, such as wherein the vector is a plasmid) from the first engineered nucleic acid (comprising the heterologous nucleic acid flanked by avian AAV ITRs ) and the second engineered nucleic acid (comprising an avian AAV rep protein gene sequence and a mammalian AAV capsid protein gene sequence) (e.g., see FIGs. 2A-2C). In some embodiments, a helper nucleic acid may be comprised on the same nucleic acid (e.g., the same vector, such as wherein the vector is a plasmid) as the first engineered nucleic acid (comprising the heterologous nucleic acid flanked by avian AAV ITRs) and / or the second engineered nucleic acid (comprising the avian AAV rep protein gene sequence and a mammalian AAV capsid protein gene sequence). 1 Regulatory Sequences

[0093] In some embodiments, engineered nucleic acids of the present disclosure comprise one or more regulatory sequences. As used herein, a “regulatory sequence” is a DNA sequence which modulates the expression, stability, and / or levels of an RNA when operably linked to a gene sequence. As used herein, a nucleic acid sequence and regulatory sequences are “operably linked” when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. For example, two DNA sequences are said to be operably linked if induction of a promoter in the 5’ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation or (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences.

[0094] Non-limiting examples of regulatory sequences that can be comprised in an engineered nucleic acid described herein include a promoter, an enhancer, a silencer, a transcription factor binding sequence, a 5' untranslated region (UTR), a 3' UTR, a translation initiation regulatory sequence, a transcriptional start sequence, a transcription termination sequence, a splicing acceptor site, a splicing donor site, a small nuclear ribonucleoprotein binding site, a mRNA degradation or decay signal, a polyadenylation signal, a Kozack sequence, a Shine-Dalgarno sequence, a start codon, a RNA-binding protein binding site, a ribosome binding site, a ribozyme, an intron, a translation termination sequence, and a stop codon. In some embodiments, an engineered nucleic acid comprises one or more regulatory sequences that regulate transcription (e.g., any one or more of a promoter, an enhancer, a transcription factor binding sequence, a transcriptional start sequence, transcription termination sequence, etc.), one or more regulatory sequences that regulate translation (e.g., any one or more of a 5’ UTR, a translation initiation regulatory sequence, a Kozack sequence, a Shine-Dalgarno sequence, a start codon, a ribosome binding site, a 3’ UTR, a translation termination sequence, a stop codon, etc.), and / or one or more regulatory sequences that regulate splicing (e.g., any one or more of a binding site for small nuclear ribonucleoproteins, splicing acceptor sites, splicing donor sites, etc.).

[0095] In some embodiments, an engineered nucleic acid described herein may comprise constitutive promoters which maintain constant expression of a gene regardless of the conditions or physiological state of a cell. Non-limiting examples of constitutive promoters include the

[0096] Herpes Simplex virus (HSV) promoter, the thymidine kinase (TK) promoter, the Simian Virus

[0097] 40 (SV40) promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (e.g., the nucleic acid sequence set forth in SEQ ID NO: 26) (optionally with the CMV enhancer) (see, e.g., disclosures related to the CMV promoter and the CMV enhancer in Boshart et al., Cell, 41:521-530 (1985)), the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerate kinase (PGK) promoter, the CAG promoter, and the human elongation factor- 1 alpha (EFla) promoter [available from, e.g., Invitrogen]. In some embodiments, a promoter comprises an RNA pol II promoter, an RNA pol III promoter, such as U6 or Hl, or an RNA pol II promoter. In some embodiments, a promoter comprises a CMV enhancer (CMVe; e.g., the nucleic acid sequence set forth in SEQ ID NO: 25). In some embodiments, a promoter comprises a chicken P-actin (CBA) promoter. In some embodiments, a promoter comprises a CMVe and a CBA promoter. In some embodiments, a promoter comprises a CAG promoter. In some embodiments, the promoter is a mouse mammary tumor virus (MMTV) promoter. In some embodiments, the promoter is a major late promoter (MLP). In some embodiments, the one or more regulatory sequences comprise an intron (e.g., an intron flanked by a splicing acceptor site and a splicing donor site). In some embodiments, the intron is a human collagen gene intron. In some embodiments, the intron is a beta globin gene intron. In some embodiments, the intron is a lambda intron.

[0098] In some embodiments, an engineered nucleic acid described herein may comprise inducible promoters. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors, such as temperature, or the presence of a specific physiological state. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters include the cytochrome P450 gene promoters, heat shock protein gene promoters, metallo thionein gene promoters, hormone- inducible gene promoters (e.g., the estrogen gene promoter zinc-inducible sheep metallothionine (MT) promoter), the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (see, e.g., disclosures related to the T7 promoter in WO 98 / 10088); the ecdysone insect promoter (see, e.g., disclosures related to the ecdysone insect promoter in No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline- repressible system (see, e.g., disclosures related to tetracycline-repressible systems in Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992), Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (see, e.g., disclosures related to RU486- inducible systems in Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (see, e.g., disclosures related to rapamycin-inducible systems in Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still, other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state.

[0099] In some embodiments, a promoter of an engineered nucleic acid described herein imparts tissue-specific gene expression capabilities. In some cases, a tissue-specific regulatory sequences bind tissue- specific transcription factors that induce transcription in a tissue specific manner. In some embodiments, a tissue-specific promoter is a neuron- specific promoter. In some embodiments, a tissue-specific promoter is a skeletal muscle- specific promoter. In some embodiments, a tissue-specific promoter is a smooth muscle-specific promoter. In some embodiments, a tissue-specific promoter is an immune cell-specific promoter. In some embodiments, a tissue-specific promoter is a cardiac muscle-specific promoter. In some embodiments, a tissue-specific promoter is a promoter that is active in a target cell that binds a pseudotyped avian rAAV particle described herein, such as a pseudotyped avian rAAV particle contacted with a target according to a method described herein (e.g., a method of treating a subject). In some embodiments, a tissue- specific promoter is a promoter that is active in a manufacturing system described herein (e.g., an isolated avian cell or an avian egg) that comprises an engineered nucleic acid described herein, such as a manufacturing system contacted with a nucleic acid comprising one or more rAAV production genes descried herein (e.g., a nucleic acid comprising either a heterologous nucleic acid flanked by avian AAV ITRs, a packaging nucleic acid, or an AAV helper nucleic acid) according to a method described herein, wherein the promoter is operably linked to the one or more rAAV production genes.

[0100] In some embodiments, a promoter of engineered nucleic acids described herein further comprise an exonic sequence. In some embodiments, a promoter further comprises an intronic sequence. In some embodiments, a promoter further comprises a chicken beta-actin intron and / or an artificial intron. In some embodiments, a promoter further comprises a chimeric intron. In some embodiments, a promoter does not comprise an exonic sequence and / or an intronic sequence.

[0101] In some embodiments, an engineered nucleic acid described herein comprises a polyadenylation (poly(A)) sequence and / or a polyadenylation (poly(A)) signal sequence inserted following a gene sequence and before any other 3' regulatory sequence (e.g., a 3' avian AAV ITR). In some embodiments, a poly(A) sequence or a polyadenylation (poly(A)) signal sequence is heterologous. In some embodiments, a poly(A) sequence is a synthetic poly(A) sequence (e.g., a non-naturally occurring poly(A) sequence that is, e.g., 20-100 nucleotides long). Examples of poly(A) signal sequences include, but are not limited to, a bovine growth hormone (bGH) poly(A) signal sequence (see, e.g., the nucleic acid sequence set forth in SEQ ID NO: 11), a SV- 40 poly (A) signal sequence, and a synthetic poly (A) signal sequences, which are known to cause polyadenylation of eukaryotic transgenes and efficient termination of translation (see, e.g., disclosures related to poly(A) signal sequences in Azzoni A R et al., J Gene Med. 2007;

[0102] 9(5):392-402). In some embodiments, a poly(A) signal is a Proudfoot poly(A) signal (see, e.g., the nucleic acid sequence set forth in SEQ ID NO: 29).

[0103] In further embodiments, other expression control elements, such as enhancer elements or introns, may also be used to regulate expression of an engineered nucleic acid (e.g., a transgene or an avian AAV rep or AAV capsid protein). In some embodiments, an engineered nucleic acid comprises one or more (e.g., 1, 2, 3, etc.) introns. In some embodiments, an intron is included in the promoter, for example, a CAG promoter or chicken beta-actin promoter. In some embodiments, an intron is included in the poly(A) signal sequence. In further embodiments, an engineered nucleic acid comprises at least one splice donor and / or splice acceptor site.

[0104] In some embodiments, at least one regulatory sequence found in an engineered nucleic acid of the present disclosure is selected from the group consisting of a promoter, an enhancer, a poly(A) signal, an intron, a Woodchuck Hepatitis Virus Post-Transcriptional Response Element (WPRE), a splicing donor / splicing acceptor element (see, e.g., the nucleic acid sequence set forth in SEQ ID NO: 27), and a bipartite leader (BPL) sequence comprising a Kozak sequence (see, e.g., the nucleic acid sequence set forth in SEQ ID NO: 7).

[0105] Vectors

[0106] Other aspects of the present disclosure relate to vectors comprising any one of the engineered acids described herein. In some embodiments, engineered nucleic acids are provided to a cell and expressed off of a vector.

[0107] In some embodiments, vectors comprise deoxyribonucleotides. In some embodiments, vectors comprise ribonucleotides. In some embodiments, vectors comprise both deoxyribonucleotides and ribonucleotides. In some embodiments, vectors are single-stranded. In some embodiments, vectors are double-stranded. In some embodiments, vectors are circular (e.g., artificial chromosomes, such as artificial bacterial chromosomes, or plasmids, such as circular plasmids, nanoplasmids, and minicircle plasmids). In some embodiments, vectors are linear. In some embodiments, vectors are self-complementary.

[0108] In some embodiments, a vector may be maintained in high levels in a cell using a selection method, such as one involving an antibiotic resistance gene. In some embodiments, a vector may comprise a partitioning sequence which ensures stable inheritance of the vector. In some embodiments, a vector is a high copy number vector. In some embodiments, a vector, or a fragment thereof (e.g., the heterologous nucleic acid), becomes integrated into the genome of a cell. In some embodiments, a vector comprises a heterologous nucleic acid flanked by avian AAV ITRs (e.g., a nucleic acid referred to herein as the “first engineered nucleic acid”) (e.g., see FIGs. IB, 3A, 11C, HE, and 11G). In some embodiments, a vector comprises an AAV packaging nucleic acid (e.g., a nucleic acid referred to herein as the “second engineered nucleic acid”) which comprises gene sequences encoding an avian AAV rep protein and an AAV capsid protein (e.g., a mammalian AAV capsid protein) (e.g., see FIGs. 3B, 11B, 11D, 11F, and 11F). In some embodiments, a vector comprises an AAV helper nucleic acid described herein (e.g., a nucleic acid referred to herein as the “third engineered nucleic acid”).

[0109] In some embodiments, a vector comprising a heterologous nucleic acid flanked by avian AAV ITRs is a recombinant viral genome (e.g., see FIGs. IB, 3A, 11C, HE, and 11G) within a pseudotyped avian rAAV particle. In some embodiments, a vector is sufficiently small to be effectively packaged in an AAV viral particle (e.g., a pseudotyped avian rAAV particle). In order to fit into the AAV viral particle (e.g., a pseudotyped avian rAAV particle), in some embodiments, an engineered nucleic acid comprising any of the heterologous nucleic acid sequences described herein comprises one or more truncated and / or recombinant sequences. Accordingly, a truncated and / or recombinant sequence is typically shorter than 4 kb but can be between around 20 bases long and around 2,000 bases long to provide space for other components (e.g., exons, regulatory sequences, other introns, viral packaging sequences) in the vector. In some embodiments, a truncated and / or recombinant sequence is shorter than 4 kb, shorter than 3 kb, or shorter than 2 kb. However, in some embodiments, a vector described herein (e.g., a plasmid) comprises 1 to 40 kb, for example from 1 to 30 kb, such as from 1 to 20 kb, for example from 1 to 15 kb, such as from 1 to 10 kb, for example from 1 to 8 kb, such as from 2 to 7 kb, for example from 3 to 6 kb, such as from 4 to 5 kb). In some embodiments, a vector (e.g., a plasmid) comprises more than 4kb (e.g., 4-10 kb, 10-25 kb, 25-50 kb, 50-75 kb, 75-100 kb, or more than 100 kb).

[0110] In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising from 5’ to 3’, in relative order, a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, the first avian AAV ITR, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, a gene sequence encoding an RNA, and a Proudfoot poly(A) signal, the second avian AAV ITR, and a bovine growth hormone (bGH) poly(A) signal. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a 5’ CELO virus ITR as set forth in the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a 3’ CELO virus ITR as set forth in the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a MLP as set forth in the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a BPL sequence as set forth in the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a Kozak ribosomal binding site as set forth in the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a first avian AAV ITR according to the nucleic acid sequence set forth in SEQ ID NO: 24. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a CMV enhancer according to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a CMV promoter according to the nucleic acid sequence set forth in SEQ ID NO: 26. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a sequence according to the nucleic acid sequence set forth in SEQ ID NO: 27 encoding a splicing donor / splicing acceptor element. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a sequence according to the nucleic acid sequence set forth in SEQ ID NO: 29 encoding a Proudfoot poly(A) signal. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a second avian AAV ITR according to the nucleic acid sequence set forth in SEQ ID NO: 30. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a bGH poly(A) signal according to the nucleic acid sequence set forth in SEQ ID NO: 11.

[0111] In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to of the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a first engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising from 5’ to 3’, in relative order, a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, the at least one gene sequence encoding an avian AAV rep protein, the at least one gene sequence encoding an AAV capsid protein, and a bovine growth hormone (bGH) poly(A) signal.

[0112] In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a 5’ CELO virus ITR of the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a 3’ CELO virus ITR of the nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a MLP of the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a BPL sequence of the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a Kozak ribosomal binding site of the nucleic acid sequence set forth in SEQ ID NO: 35. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a sequence according to SEQ ID NO: 9 encoding an avian AAV rep protein. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a sequence according to SEQ ID NO: 30 encoding an AAV capsid protein. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a bGH poly(A) signal according to the nucleic acid sequence set forth in SEQ ID NO: 11.

[0113] In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of the nucleotide sequences set forth in SEQ ID NOs: 1, 15, 17, 19, or 32. In some embodiments, a second engineered nucleic acid of a plurality of nucleic acids described herein is a vector comprising any one of the nucleic acid sequence set forth in SEQ ID NOs: 1, 15, 17, 19, or 32.

[0114] In some embodiments, a vector that may be engineered to produce nucleic acids used for producing a pseudotyped avian rAAV particle comprises from 5’ to 3’, in relative order, a gene sequence encoding an avian AAV rep protein, and a gene sequence encoding an AAV capsid protein, wherein the vector further comprises a 5’ CELO virus ITR and a 3’ CELO virus ITR. In some embodiments, a vector that may be engineered to produce nucleic acids used for producing a pseudotyped avian rAAV particle comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 34. In some embodiments, a vector that may be engineered to produce nucleic acids used for producing a pseudotyped avian rAAV particle comprises the nucleic acid sequence set forth in SEQ ID NO: 34.

[0115] Examples of Engineered Nucleic Acid Sequences

[0116] Tables 1-4 and 6-7 provide non-limiting examples of vectors that can be used to produce pseudotyped AAV particles as described herein. Table 5 provides a non-limiting example of a packaging nucleic acid comprising an avian AAV cap gene. Table 8 provides a non-limiting example of a vector comprising sequences that may be used a backbone for engineering vectors for pseudotyped rAAV particle production. Table 9 provides a non-limiting example of a helper nucleic acid. One or more of the packaging genes, helper genes, and / or regulatory elements described in the following examples of vectors can be used alone or in combination with other packaging genes, helper genes, and / or regulatory elements.

[0117] Table 1: Sequences of PAdCEV- Avian- AAV-Rep-AAV6-Cap

[0118] Table 2: Sequences of PAdCEV- Avian- AAV-Rep-AAV7-Cap

[0119] Table 3: Sequences of PAdCEV-Avian-AAV-Rep-AAV8-M3-Cap

[0120] Table 4: Sequences of PAdCEV- Avian- AAV-Rep-AAV9-Cap

[0121] Table 5: Sequences of PAdCEV- Avian- AAV-Rep- Avian-Cap

[0122] Table 6: Sequences of pAdCEV-Avian-ITR-CMV-hGFP

[0123]

[0124]

[0125] Table 7: Sequences of pAdCEV-Avian-Rep-AAVl-Cap

[0126] Table 8: Sequences of pAfriVaxl

[0127]

[0128] Table 9: Sequence of pBeloBacll-CELO-gDNA-wt

[0129] In some embodiments, the present disclosure embraces engineered nucleic acids of a given % identity to any one of the nucleic acid sequences set forth in a SEQ ID NO provided herein. Accordingly, the present disclosure encompasses both variant forms of engineered nucleic acids corresponding to the nucleic acid sequence set forth in a SEQ ID NO comprising different vector- specific sequences (e.g., different vector backbone sequences) and / or those with different sequences of heterologous nucleic acids, ITRs, AAV capsid proteins, etc.

[0130] The term “% sequence identity” or “percentage sequence identity” with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a query sequence that are identical with the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the means of one of ordinary skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (see, e.g., disclosures related to sequence alignments in Ausubel et al., eds., 1987, Supp. 30, section 7.7.18, Table 7.7.1), and including, but not limited to, BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), COBALT, OPAL, Multlin, Clustal Omega, Clustal W2.0, or Clustal X2.0 software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the nucleic acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (e.g., a segment of nucleic acids that is used for alignment purposes against a query sequence which does not include additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleotide, nucleoside, or nucleobase occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence.

[0131] Accordingly, it should be recognized that, while examples of engineered nucleic acids (e.g., the “first engineered nucleic acid”, such as an rAAV genome; the “second engineered nucleic acid”, such as a packaging nucleic acid; the helper nucleic acid, such as one provided on the second engineered nucleic acid or as a “third engineered nucleic acid”; etc.) are described herein (e.g., see Tables 1-4 and 7-9), any nucleic acid (e.g., an engineered nucleic acid) for production of pseudotyped avian rAAV particles is provided by the present disclosure. In some embodiments, such a nucleic may comprise a substitution, addition, or deletion of at least one nucleotide relative to any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-20 and 23-36. In some embodiments, such a nucleic acid may comprise a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-40, 40-50, 50-100, 100-250, 250-500, 500-1000, 1000-2000, 2000- 3000, 3000-4000, etc.) of nucleotide positions which differ (e.g., nucleotide positions comprising a substitution, addition, or deletion of a nucleotide) relative to any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-20 and 23-36. In some embodiments, such a nucleic acid may comprise at least 75% or more (e.g., 80-85%, 85-90%, 90-95%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 1- 20 and 23-36. In some embodiments, such a nucleic acid may comprise a sequence encoding: the avian AAV ITRs flanking a heterologous nucleic acid; an avian AAV rep gene sequence; or a helper nucleic acid; wherein the sequence is set forth in any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-20 and 23-36, but further comprises a backbone sequence (e.g., a vector backbone sequence) which differs from the backbone sequence any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-20 and 23-36 have been cloned into. In some embodiments, any of such nucleic acids may comprise a vector.

[0132] For example, in some embodiments, such a nucleic acid may comprise a heterologous nucleic acid flanked by avian AAV ITRs but does not comprise one or more of a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, a gene sequence encoding an RNA, a Proudfoot poly(A) signal, the second avian AAV ITR, and a bovine growth hormone (bGH) poly(A) signal. In some embodiments, such a nucleic acid may be a recombinant genome of a pseudotyped avian rAAV particle. As a further example, in some embodiments, such a nucleic acid may comprise an avian AAV rep gene and a non-avian AAV cap gene but does not comprise a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, or a bovine growth hormone (bGH) poly(A) signal. In some embodiments, such a nucleic acid may comprise a packaging nucleic acid. Moreover, in some embodiments, such as a nucleic acid may comprise a portion of a CELO virus nucleic acid sequence described herein. In some embodiments, such a nucleic acid may be provided on the same nucleic acid molecule as the packaging nucleic acid comprising the avian AAV rep gene or it may be a separate molecule. In some embodiments, such a nucleic acid may comprise a helper nucleic acid. In some embodiments, any of the above-described variations on the engineered nucleic acids of Tables 1- 4 and 6-9 may be isolated nucleic acids (e.g., not in a cell or manufacturing system) or may be in a manufacturing system (e.g., a cultured cell, such as an avian cell, or an avian egg, such as an embryonated egg). In some embodiments, any of the above-described variations on the engineered nucleic acids of Tables 1-4 and 6-9 may be provided as a single nucleic acid (e.g., as a single molecule or as a plurality of nucleic acid species provided in a homogenous mixture) or as a mixture of more than one nucleic acid species (e.g., a heterogenous mixture of rAAV genomes and packaging nucleic acids).

[0133] Manufacturing Systems

[0134] As used herein, the term “manufacturing system” refers to a biological host system that is capable of both packaging and propagation of an rAAV particle (e.g., pseudotyped avian rAAV particles). Manufacturing systems include both non-natural (e.g., host cells, such as avian cell lines ) and natural (e.g., avian eggs, such as embryonated eggs) systems described herein. In some embodiments, a manufacturing system is generated by engineering a host cell (e.g., an avian cell, such as a cell in avian egg including, but not limited to, embryonated eggs) to comprise a nucleic acid described herein. In some embodiments, the nucleic acid comprise a heterologous nucleic acid flanked by avian AAV ITRs. In some embodiments, the nucleic acid comprises a packaging nucleic acid (e.g., a nucleic acid comprising AAV rep and / or AAV cap genes). In some embodiments, the nucleic acid comprises a helper nucleic acid (e.g., a nucleic acid comprising an El gene, an E2A gene, an E4 gene, a VA gene, or a combination thereof). In some embodiments, the nucleic acid is delivered to a manufacturing system (e.g., an avian cell, such as a cell in avian egg including, but not limited to, embryonated eggs) by contacting the manufacturing system with a vector described herein (e.g., a plasmid). Manufacturing systems of the present disclosure are suited for high yield production of pseudotyped avian rAAV particles.

[0135] In some embodiments, methods and manufacturing systems for production of pseudotyped avian rAAV particles comprise use of a helper nucleic acid. In some embodiments, the helper nucleic acid comprises a plurality of AAV helper genes described herein, such as two, three, four, five, six, seven, eight, nine, ten, or more than ten AAV helper genes. In some embodiments, the helper nucleic acid comprises at least one of an El gene, an E2A gene, an E4 gene, or a VA gene. In some embodiments, manufacturing systems comprise an adenovirus helper nucleic acid, such as an avian adenovirus helper nucleic acid. In some embodiments, the avian adenovirus helper nucleic acid is a CELO helper virus nucleic acid. In some embodiments, the helper virus nucleic acid is provided on a vector (e.g., a plasmid). In some embodiments, the helper nucleic acid comprises a sequence with least 75% identity (e.g., 75-80%, 80-85%, 85- 90%, 90-95%, or 95-100% identity) to a nucleotide sequence comprised in SEQ ID NO: 36 (e.g., at least 75% identity to a helper sequence comprised in the nucleotide sequence set forth in SEQ ID NO: 36, such as a helper sequence comprising at least one of El, E2A, E4, or VA genes).

[0136] In some embodiments, manufacturing systems are transduced with a helper virus, or a recombinant version thereof, comprising a helper nucleic acid described herein. In some embodiments, manufacturing systems are transduced with a helper virus comprising at least one of an El gene, an E2A gene, an E4 gene, or a VA gene. In some embodiments, a helper virus, or a recombinant version thereof which is contacted with a manufacturing system described herein is not a herpes simplex virus (HSV) or a recombinant HSV.

[0137] In some embodiments, manufacturing systems are transduced with an adenovirus which provides AAV helper functions. In some embodiments, manufacturing systems are transduced with an avian adenovirus which provides AAV helper functions. In some embodiments, manufacturing systems are transduced with a CELO virus which provides helper functions. In some embodiments, manufacturing systems are transduced with a helper nucleic acid comprising a sequence with least 75% identity to SEQ ID NO: 36.

[0138] In some embodiments, methods and manufacturing systems for production of pseudotyped avian rAAV particles comprise use of a packaging nucleic acid. In some embodiments, the packaging nucleic acid comprises an avian AAV rep protein gene sequence and an AAV capsid protein gene sequence (e.g., avian AAV capsid protein gene sequences or mammalian AAV capsid protein gene sequences). In some embodiments, wherein the packaging nucleic acid comprises avian AAV rep and mammalian AAV capsid protein gene sequences, the heterologous nucleic acid is flanked by avian AAV ITRs. In some embodiments, the packaging nucleic acid is a vector.

[0139] In some embodiments, helper nucleic acids and / or packaging nucleic acids are provided in trans to a manufacturing system. In some embodiments, manufacturing systems are engineered to stably express helper nucleic acids and / or packaging nucleic acids.

[0140] Host Cells

[0141] In some embodiments, a host cell is contacted with one or more engineered nucleic acids or composition thereof described herein in order to produce pseudotyped avian rAAV particles. As used herein, a “host cell” refers to a cell that is used for the production of AAV and rAAV particles and is transduced and / or engineered in vitro with the required nucleic acid constructs described herein in order to manufacture said particles. In some embodiments, a host cell is stably transfected with a heterologous nucleic acid flanked by avian AAV ITRs. In some embodiments, a host cell is stably transfected with the at least one of the helper nucleic acid genes and / or the avian AAV rep and AAV capsid protein genes of the second engineered nucleic acid (e.g., see FIG 2B).

[0142] In some embodiments, a host cell is an avian cell. In some embodiments, the host cell is a chicken cell. In some embodiments, the chicken cell is a EB14 cell, an HD-11 cell, a DF-1 cell, a DT95 cell, a DT40 cell, a LMH cell, or a PBS- 12 SF cell. In some embodiments, the host cell is a duck cell. In some embodiments, the duck cell is an AGE1.CR cell, an AGEl.CR.pIX cell, an EB66 cell, or a DEF cell. In some embodiments, the host cell is a quail cell. In some embodiments, the quail cell is a QEF cell, a QM7 cell, a QT-35 cell, or a QT-6 cell. In some embodiments, the host cells is a goose cell. In some embodiments, a host cell is an avian cell described herein which is an ex vivo avian cell. In some embodiments, the host cell is a suspension cell. In some embodiments, the host cell is a 293 cell or HEK293 cell, a HEK293T cell, COS cells, HeLa cells, HeLaS3 cells, BHK cells, CHO cells or PER.C6® cells (see, e.g., ATCC® CRL-1573™, ATCC® CRL-1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL- 2.2, ATCC® CCL-10™, or ATCC® CCL-61™).

[0143] Avian Eggs

[0144] In some embodiments, avian eggs are used as manufacturing systems for pseudotyped rAAV particle production. In some embodiments, avian eggs used as manufacturing systems for pseudotyped avian rAAV particles are embryonated avian eggs. As used herein, the term “embryonated avian egg” refers to a fertilized egg of an avian species in which an embryo has formed, wherein the egg contains an allantoic cavity, an amnion, and a yolk sac. The disclosure embraces avian eggs of various species, such as chicken eggs, duck eggs, quail eggs, and goose eggs. In certain embodiments, the egg to be inoculated is 10 days old, as determined by candling. In other embodiments, the egg to be inoculated is 8 days old, 9 days, old, 11 days old, 12 days old, 13 days old, or 15 days old.

[0145] In some embodiments, an avian egg (e.g., an embryonated egg) is contacted with one or more engineered nucleic acids or composition thereof described herein.

[0146] Compositions

[0147] Compositions of the present disclosure include both compositions for rAAV production, (e.g., pseudotyped avian rAAV particle production) (e.g., compositions that comprise one or more engineered nucleic acids (e.g., a plurality of engineered nucleic acids) which are required for viral particle production as described herein and optionally a host cell or population thereof) and pharmaceutical compositions comprising pseudotyped avian rAAV particles described herein.

[0148] Pharmaceutical Compositions and Ingredients Thereof

[0149] "Pharmaceutical compositions” of the present disclosure comprise pseudotyped avian rAAV particles and at least one other pharmaceutically acceptable ingredient described herein, wherein the pseudotyped avian rAAV particles are produced by any suitable method.

[0150] In some embodiments, a composition comprising a pseudotyped avian rAAV particle comprises a liposome, a lipid, a lipid complex, a lipid nanoparticle, a microsphere, a microparticle, a nanosphere, and / or a nanoparticle, or may be otherwise formulated for administration to the cells, biological samples, tissues, organs, or body of a subject in need thereof.

[0151] In some embodiments, a composition comprising a pseudotyped avian rAAV particle comprises a pharmaceutical excipient. Pharmaceutically acceptable excipients (excipients) are substances other than a therapeutic agent that are intentionally included in a drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute of the overall safety, effectiveness, or delivery of the therapeutic agent during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance. Excipients include, but are not limited to, absorption enhancers, anti- adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.

[0152] In some embodiments, a pharmaceutical composition comprising a pseudotyped avian rAAV particle comprises additional components commonly found in pharmaceutical compositions. Such additional components can include, but are not limited to: anti-pruritic s, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine) .

[0153] In some embodiments, pharmaceutical compositions of the present disclosure may be suitable for treatment regimens and thereby administered to a subject via a variety of methods described herein. Such compositions may be formulated for use in a variety of therapies, such as, for example, in the amelioration, prevention, and / or treatment of conditions. Accordingly, compositions described herein may be administered to a subject, such as human or non-human subjects, a host cell in situ in a subject, a host cell ex vivo, a host cell derived from a subject, or a biological sample (e.g., one derived from a subject).

[0154] For administration of an injectable aqueous solution, the composition may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline, polyalcohols, or glucose. For example, one dosage of a therapeutic agent may be dissolved in an isotonic NaCl solution and optionally added to a larger volume of hypodermoclysis fluid prior to being injected at the proposed site of infusion. In some embodiments, the composition is provided in a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. A composition may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0155] Methods

[0156] Methods of Producing Pseudotyped Avian Recombinant Adeno-Associated Virus Particles

[0157] Aspects of the present disclosure relate to methods of packaging AAV vectors in avian manufacturing systems (e.g., avian cells, such as ex vivo avian cells, and avian eggs, such as embryonated chicken eggs). Methods of producing rAAV particles (e.g., pseudotyped avian rAAV particles) in avian eggs, including embryonated eggs, have been previously described (e.g., see, rAAV particle production methods described in PCT / US2020 / 048632, published as International Publication No. WO 2021 / 041986, which is incorporated by herein in its entirety). Non-limiting examples of pseudotyped avian rAAV particle production methods are described in FIGs. 2A-2C, 4, 5, 6B, 7A-7B, 8A-8C, and 9. In some embodiments, packaging comprises an inoculation step. In some embodiments, packaging comprises a transfection method. The described methods may make use of one or more nucleic acid vectors, such as two or more plasmids (e.g., packaging and helper nucleic acids). Alternatively, they may make use of one or more virus particles, such as two virus particles, to deliver one or more genes for pseudotyped avian rAAV particle production. In some embodiments, the one or more virus particles do not comprise a herpes simplex virus (HSV) particle. In some embodiments, the disclosed methods make use of one or more helper virus particles described herein (e.g., adenovirus, such as avian adenovirus). In some embodiments, the disclosed methods make use of one or more CELO helper virus particles. In some embodiments, the produced AAV is a pseudotyped avian rAAV particle described herein.

[0158] Accordingly, the disclosed methods of producing pseudotyped avian rAAV particles, in some embodiments, comprise: i) inoculating an avian egg with the first engineered nucleic acid (e.g., a vector comprising a heterologous nucleic acid) and the second engineered nucleic acid (e.g., a vector comprising a gene sequence encoding one or more avian AAV rep proteins and a gene sequence encoding one or more AAV capsid proteins), ii) incubating the egg, and iii) isolating rAAV virions (or particles) from the egg. Inoculation and Infection Methods

[0159] AAV genes and any genes desired to be packaged into pseudotyped avian rAAV particles may be introduced to cells by either transfection methods (e.g., using vectors and a transfection agent) or infection methods (e.g., using a viral vector). Cells are said to be “transfected” or “infected” at the time when transfection or infection reagents (e.g., vectors) are first introduced to the cells.

[0160] In some embodiments, an allantoic cavity, a chorioallantoic membrane, a yolk sac, or an amnion of the egg is inoculated (FIG. 6A) with one or more nucleic acids described herein. The size of the allantoic fluid is related to the stage of embryonic development of the egg to be injected; thus, the depth of injection or insertion needed to reach the allantoic fluid will vary depending on the developmental stage of the egg as well as the species and strain of avian egg used. The depth of injection or insertion must be deep enough to place the needle or probe within the allantoic fluid, but not so deep as to pierce the amnion or embryo. Use of a blunt- tip needle helps minimize piercing of the amnion or embryo. In chicken eggs at days 7-15 of embryonation, injection or insertion from l / 8thto 14thof an inch below the egg shell surface is preferred. Examples of injection methods are provided in FIG. 6B. Age of embryonation may be determined by candling the egg. For example, see FIG. 6C.

[0161] In some embodiments, inoculation comprises manual injection. An example of a manual injection method is provided in FIG. 7B. In some embodiments, inoculation comprises automatic injection. Examples of automatic injection methods are provided in FIGs. 8A-8C. Exemplary automatic egg injectors suitable for use in the disclosed methods of propagating AAV in avian eggs include, but are not limited to, injectors manufactured by Sanovax. These injectors have demonstrated suitability for use in growth of influenza vaccine in embryonated chicken eggs. A number of automatic egg injection devices have been developed. These include devices described in U.S. Pat. No. 5,056,464 to Lewis: U.S. Pat. No. 4,903,635 and U.S. Pat. No. 4,681,063 to Hebrank; U.S. Pat. No. 5,136,979 to Paul et al.: and U.S. Pat. Nos. 4,040,388, 4,469.047 and 4,593,646 to Miller, each of which are incorporated herein by reference. Automated injection methods of the disclosure may involve delivering helper virus particles or AAV vectors in fluid form to the interior of an egg using an automated machine which delivers the vaccine to the egg through a needle. The needle can be used to both penetrate the egg shell and deliver the fluid substances, or the opening in the shell can be performed separately in advance of the fluid injection. The egg can be injected at any location within the egg, such as the allantoic fluid or through the CAM. In some embodiments, methods of pseudotyped avian rAAV particle production in avian eggs include inoculation through the uppermost side of the egg. Many commercial egg injection apparatus are designed to inject eggs using a needle that is vertical and that travels downward into the egg. See e.g., disclosures related to egg injection in: U.S. Pat. No. 4,469,047 to Miller; U.S. Pat. No. 4.681,063 to Hebrank; U.S. Pat. No. 4,903,635 to Hebrank; U.S. Pat. No. 5,056,464 to Lewis; U.S. Pat. No. 5,136,979 to Paul and Ilich; and published PCT application WO 98 / 31216 to Bounds, each of which are incorporated herein by reference. The disclosed methods comprise methods of orienting eggs also allow vertical downward injection while avoiding piercing the air cell membrane. Accordingly, a preferred method of injection is downward along the path of the needle since this method of injection is more readily accomplished with minimum modification to existing automatic injection machines.

[0162] It will be appreciated by those skilled in the art that the precise location and angle of injection is a matter of choice and could be in any area of the egg’s upper surface that overlies the allantoic fluid. Orientation of the needle will depend on the orientation of the egg and the equipment available to carry out the injection. While the orientation of the egg may be about 45 degrees from vertical, the orientation may extend from about 10 degrees up to 180 degrees. Preferred angles of egg orientation include from about 20 degrees to about 45 degrees. Injecting the egg with the long axis of the egg more nearly vertical, e.g., an angle of less than about 10 degrees, increases the chance that the injection needle will traverse the air cell. Where it is desired to avoid piercing the air cell, routine experimentation using eggs of similar age and condition and from the same breed and strain of bird, will allow determination of the minimum angle of egg orientation needed to avoid piercing the air cell in a majority of such eggs.

[0163] Automated machines and methods for simultaneously injecting a large number of eggs are known. In one well known commercial machine, the eggs in the incubating trays are brought under a bank of injectors which house both needles and punches. First, the punches open a hole in the egg shell. Then, the needle is inserted into the egg through the open hole, followed by injection of the fluid. The punch is necessary because the needle is long and thin and cannot repeatedly punch egg shells without bending and / or clogging. This system is described, for example, in U.S. Pat. No. 4,691,063 to Hebrank. In another machine, such as that described in U.S. Pat. No. 6,240,877 Bl, the injectors house a single needle which both punches the hole in the egg shell with a closed needle end and then delivers the fluid through a hole in the side of the needle tip.

[0164] In certain embodiments, the avian egg is candled prior to inoculation and / or prior to harvesting. As used herein, “candling” refers to the process of holding the egg, or parts of the egg, in front of a light source, such as a candle, light bulb or fluorescent light source, to determine the stage of development of the embryo. Candling may reveal whether the embryo is alive or viable. In certain embodiments of the disclosed methods, the first and second engineered nucleic acid vectors are transfected with a cationic polymer (e.g., polyethyleneimine) prior to inoculation.

[0165] Incubation Methods

[0166] In some embodiments, the inoculated egg is incubated in a shaker, a spinner, or an automatic egg incubator. In some embodiments, the inoculated AEF cultures are incubated in a shaker flask, a spinner flask, a cellbag, or a bioreactor.

[0167] In some embodiments, the egg is incubated after inoculation for a period of at least 15 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 80 hours, at least 90 hours, or at least 96 hours. In some embodiments, the egg may be incubated for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 15 days. In some embodiments, the egg is incubated for about 7 days.

[0168] At about the beginning of the final quarter of incubation, the eggs are transferred from the incubator to a hatcher. This step is known as “transfer”. At transfer, the step of sampling or sensing the allantoic fluid to assess the condition or status of the embryo within the egg may advantageously be carried out, as can injection of beneficial substances into the allantoic fluid. An egg tray may be used in automatic egg injection equipment. The egg tray comprises a base containing a plurality of egg receptacles which are configured to hold the eggs at a desired angle. Typically, eggs are incubated in an incubating tray placed in an incubator or setter machine. Conventional incubating trays include the Chick Master® 54 tray, the Jamesway® 42 tray, and the Jamesway® 84 tray (in each case, the number indicates the number of eggs carried by the tray). The eggs from three Chick Master® 54 trays, or a total of 162 eggs, would be transferred to a single hatcher tray; the eggs from four Jamesway® 42 trays, or a total of 168 eggs, would be transferred to a single hatcher tray; and the eggs from two Jamesway® 84 trays, or a total of 168 eggs, would be transferred to a single hatcher tray. Some incubating trays, such as the La Nationale® incubating tray, which are sufficiently large enough to include a total number of eggs, in this case 132 eggs, such that the eggs from a single incubating tray would be transferred to a corresponding hatcher tray.

[0169] Isolation, Purification, and Propagation Methods In some embodiments, the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) is substantially free of avian viruses. In some embodiments, the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) is isolated from the allantoic fluid. In some embodiments, the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) is isolated from the yolk. In some embodiments, the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) is isolated from the embryo. In some embodiments, the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) is isolated from the whole egg or a plurality of components of the egg (e.g., isolated from two or more components selected from: the allantoic fluid, the yolk, and the embryo). An example of manual isolation for harvesting allantoic fluid are provided in FIGs. 7C and 9. In some embodiments, the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) is isolated using a manual pipette, a manual syringe, a machine-controlled pipette, or a machine-controlled syringe. In some embodiments, the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) or purified rAAV particle (e.g., purified pseudotyped avian rAAV particle) is added to a pharmaceutical composition.

[0170] In some embodiments, the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) or purified rAAV particle (e.g., purified pseudotyped avian rAAV particle) is at a titer of at least about 1 x 108, at least about 5 x 108, at least about 1 x 109, at least about 5 x 109, at least about 1 x IO10, at least about 5 x IO10, at least about 1 x 1011, at least 2 x 1011, or at least 5 x 1011vector genomes (vg) / ml. AAV vector genome concentrations (or titers) may be measured by any method known in the art. Exemplary methods include quantifiable polymerase chain reaction (qPCR). In some embodiments, following purification steps, a volume of about 200 pL of pure AAV vector per egg may be recovered. In other embodiments, other volumes may be obtained including, but not limited to, about 50 pL, 75 pL, 100 pL, 150 pL, 175 pL, 180 pL, 190 pL, 210 pL, 220 pL, 225 pL, 250 pL, or 300 pL of pure AAV vector per egg may be recovered.

[0171] In some embodiments, the method further comprises a purification step. In particular embodiments, the methods comprise subjecting the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) to an iodixanol gradient and / or affinity chromatography (see, e.g., FIG. 5). A discontinuous iodixanol gradient (e.g., a gradient of between 15% and 54%) may be used for these methods. In some embodiments, an iodixanol gradient is used wherein the lowest concentration of iodixanol in the gradient is 15%. In some embodiments, the methods comprise subjecting the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) to ion exchange chromatography. Additional affinity purification methods that may be used with the rAAV virions of this disclosure are disclosed in U.S. Publication No. 2017 / 0130208, herein incorporated by reference. The resulting separated mixture may be centrifuged (e.g., at 350,000g) for about 1 hour to isolate rAAV (e.g., pseudotyped avian rAAV). In some embodiments, purification comprises contacting the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) with an affinity resin. In some embodiments, purification comprises subjecting the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) to iodixanol gradient ultracentrifugation and contacting the isolated rAAV particle (e.g., isolated pseudotyped avian rAAV particle) with an affinity resin. Tangential flow filtration (TFF), dialysis membrane filtration, and / or centrifugation (e.g., using centrifugation filtration devices, e.g., at 150kD Molecular weight cut-off (MWCO) membrane filter devices, see, e.g., products from Orbital Bioscience and Millipore). In some embodiments, the affinity resin may comprise the POROS™ CaptureSelect™ AAVX Affinity Resin. Other examples of purification / concentration approaches involve commercially available TFF systems and cartridges include products from GE Healthcare Life Sciences (see, e.g., the Midgee products) 5 and Pall Corporation (see, e.g., Minimate™ TFF System).

[0172] In some embodiments of methods described herein, the methods may further comprise measuring the purity and / or the amount of intact rAAV particles in the purified rAAV particles (e.g., purified pseudotyped avian rAAV particles). Measuring the purity and / or the amount of intact rAAV particles in the purified rAAV particles (e.g., purified pseudotyped avian rAAV particles) may be accomplished using any method known in the art. In some embodiments, measuring the purity and / or the amount of intact rAAV particles (e.g., pseudotyped avian rAAV particles) comprises an immunoassay, a nucleic acid hybridization-based assay (e.g., a dot-blot assay), SDS-PAGE followed by either Coomassie blue or silver staining, visualization with an electron microscope, a PCR assay, an infectious center assay (e.g., green fluorescent cell assay), or combinations thereof. In some embodiments, measuring the purity and / or the amount of intact rAAV particles (e.g., pseudotyped avian rAAV particles) comprises an immunoassay that includes antibodies specific for intact capsids.

[0173] In some embodiments, the methods may subsequently comprise inoculating an avian egg or host cells with the purified rAAV (e.g., pseudotyped avian rAAV), and propagating the rAAV (e.g., pseudotyped avian rAAV) by incubating the avian egg (e.g., chicken egg) or host cells, and isolating the rAAV (e.g., pseudotyped avian rAAV). In some embodiments, the chorioallantoic membrane (CAM) of the egg is inoculated. In other embodiments, the allantoic cavity of the egg is inoculated. Recombinant AAV particles (e.g., pseudotyped avian rAAV particles) isolated from a first avian egg as above may be propagated in a second avian egg for larger scale growth. In some embodiments, the first avian egg (for packaging) and second avian egg (for propagation) may be isolated from the same “batch” of avian eggs, as handled by an automatic egg handler (or injector). Alternatively, in some embodiments, the first avian egg may be isolated from a different batch of eggs than the second avian egg. In some embodiments, the rAAV (e.g., pseudotyped avian rAAV) is further propagated in mammalian cells or insect cells. In some embodiments, mammalian cells may be, for example, HEK293 cells, baby hamster kidney (BHK) cells, or HeLa cells. In some embodiments, insect cells may be, for example, Sf9 cells. In other embodiments, AEF (e.g., CEF) in culture is used as a host vehicle for propagation of AAV vectors. It should be appreciated that any cell or cell line that is known in the art to propagate rAAV particles can be used in embodiments of the methods disclosed herein, following the packaging of rAAV in avian eggs. In some embodiments, a cell used to propagate rAAV (e.g., pseudotyped avian rAAV) in the methods disclosed herein is a mammalian cell. Non-limiting examples of mammalian cells that can be used to propagate rAAV (e.g., pseudotyped avian rAAV) are 293 cells, HEK293 cells, HEK293T cells, COS cells, HeLa cells, HeLaS3 cells, BHK cells, CHO cells or PER.C6® cells (see, e.g., ATCC® CRL-1573™, ATCC® CRL- 1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-2.2, ATCC® CCL-10™, or ATCC® CCL-61™). In some embodiments, a cell used to propagate rAAV particles (e.g., pseudotyped avian rAAV particles) is an insect cell. An example of insect cells includes Sf9 cells (see, e.g., ATCC® CRL-1711™). In some embodiments, Sf9 cells may be Sf9 producer cells that comprise rep and / or capsid protein genes from one or more AAV serotypes or pseudotypes for producing rAAV particles (see, e.g., disclosures related to AAV production in: Mietzsch et al. OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1-12 vectors for gene therapy. Hum Gene Ther. 2014 Mar;25(3):212-22; and Aslanidi et al. An inducible system for highly efficient production of recombinant adeno- associated virus (rAAV) vectors in insect Sf9 cells. Proc Natl Acad Sci U S A 2009 106: 5059- 5064). In some embodiments, a cell used to propagate an rAAV is an avian cell.

[0174] In some embodiments, production methods comprise propagating rAAV particles (e.g., pseudotyped avian rAAV particles) in avian eggs wherein the rAAV may have been packaged in a vehicle other than avian eggs, such as baculovirus. In these methods, avian eggs (e.g., chicken eggs) are used as a host vehicle for propagation of AAV vectors. In other aspects, the disclosed production methods comprise propagating rAAV particles (e.g., pseudotyped avian rAAV particles) in host cells wherein the rAAV may have been packaged in a vehicle other than avian eggs, such as baculovirus.

[0175] Methods of Treatment

[0176] In other aspects, the present disclosure relates to methods of treating or preventing a disease, disorder, or condition in a subject using any of the pseudotyped avian rAAV particles or compositions thereof described herein.

[0177] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0178] A “subject” to which administration is contemplated refers to a human (e.g., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal. The term “patient” refers to a human subject in need of treatment of a disease. The term “subject in need thereof’ refers to a human subject having, at risk of having, previously had, or is suspected of having a disease or disorder.

[0179] In some embodiments, treating a subject in need thereof using any of the methods of the present disclosure comprises administering a therapeutically effective amount of a pseudotyped avian rAAV particle or composition thereof described herein. A “therapeutically effective amount” of a therapeutic agent described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent.

[0180] In some embodiments, administration of a pseudotyped avian rAAV particle or composition thereof described herein achieves one, two, three, four, or more of the following effects, including, for example: (i) reduction or amelioration the severity of disease, disorder, or condition or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease, disorder, or condition; (iii) protection against the progression of a disease or disorder or symptom associated therewith; (iv) regression of a disease, disorder, or condition or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease, disorder, or condition; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease, disorder, or condition; (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.

[0181] In certain circumstances, it will be desirable to perform administration of a pseudotyped avian rAAV particle or composition thereof described herein subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intracranially, intrathecally, orally, intraperitoneally, or by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs. In some embodiments, direct injection is performed concurrently with a surgical procedure or interventional procedure. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent, and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration, injection, etc.). In some embodiments, compositions are administered to a subject through only one administration route. In some embodiments, multiple administration routes may be exploited (e.g., serially, or simultaneously) for administration of the composition to a subject. Treatment methods involving rAAV particles are also known in the art (see, e.g., disclosures related to rAAV administration in: Adeno-Associated Virus Vectors in Clinical Trials. Barrie J. Carter. Human Gene Therapy. May 2005, 16(5): 541-550. doi: 10.1089 / hum.2005.16.541. Published in Volume: 16 Issue 5: May 25, 2005; Neuropharmacology. 2013 Jun;69:82-8. doi: 10.1016 / j.neuropharm.2012.03.004. Epub 2012 Mar 17.; Adeno-associated virus (AAV) gene therapy for neurological disease. Weinberg MSI, Samulski RJ, McCown TJ.Gene therapy for lysosomal storage disorders. Yew NS, Cheng SH. Pediatr Endocrinol Rev. 2013 Nov;l l Suppl 1:99-109; Directed evolution of novel adeno- associated viruses for therapeutic gene delivery. Bartel MA, Weinstein JR, Schaffer DV.Gene Ther. 2012 Jun;19(6):694-700. doi: 10.1038 / gt.2012.20. Epub 2012 Mar 8; Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Mingozzi F, High KA. Nat Rev Genet. 2011 May;12(5):341-55. doi: 10.1038 / nrg2988).

[0182] In some embodiments, the subject has, is suspected of having, or at risk of developing a disease, disorder, or condition. In some embodiments, the subject is a human. In some embodiments, the disease is a genetic disease arising from a single mutated gene (e.g., a monogenic disease). In some embodiments, the disease is a genetic disease arising from multiple mutated genes (e.g., a polygenic disease comprising two, three, or more mutated genes). In some embodiments, the disease involves toxicity symptoms in patients upon administration of a therapy delivered using non-pseudotyped rAAV particles (e.g., AAV2-dependent toxicity as seen in Aromatic L- Amino Acid Decarboxylase Deficiency, Lever Congenital Amaurosis Disease, Duchenne Muscular Dystrophy (DMD), Hemophilia, Spinal Muscular Atrophy (SMA), Friedreich’s Ataxia, Pompe Disease, Alpha- 1 Antitrypsin Deficiency, X-linked Severe Combinate Immunodeficiency Disease (SCID), and Cystic Fibrosis). In some embodiments, the disease, disorder, or condition comprises a genetic disease, cancer, inflammatory disease or a inflammatory condition, autoimmune disease, liver disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, a kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or other disease, or any combination thereof. In some embodiments, pseudotyped avian rAAV particles may be used for the purposes of vaccinating a subject against infection of a pathogen. Non-limiting examples of pathogens include a virus of the family of Adenoviridae, Picomaviridae, Herpesviridae, Hepadnaviridae, Coronaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Poxviridae, Rhabdoviridae, and Togaviridae, a bacteria such as Mycobacterium tuberculosis, Streptococcus, Pseudomonas, Shigella, Campylobacter, and Salmonella, or a fungus species such as Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocytis, Stachybotrus, Bacillus anthracis, Clostridium botulinum, Mycobacterium leprae, Yersinia pestis, Rickettsia prowazekii, Bartonella spp., or another organism, such as a parasite that causes malaria, amoebiasis, babesiosis, giardiasis, toxoplasmosis, cryptosporidiosis, trichomoniasis, Chagas disease, leishmaniasis, African trypanosomiasis (sleeping sickness), Acanthamoeba keratitis, and primary amoebic meningoencephalitis (naegleriasis). In some aspects, the disclosure relates to kits comprising any of the engineered nucleic acids, pseudotyped avian rAAV particles, host cells, avian eggs (e.g., embryonated avian eggs), and / or compositions described herein. In some embodiments, the kits described herein may be used, for producing a pseudotyped avian rAAV particle or treatment of a subject in need thereof according to any of the methods described herein.

[0183] In some embodiments, the kits described herein may include one or more containers housing components for performing the methods described herein, and optionally instructions for use. In some embodiments, components may be prepared sterilely, packaged in a syringe, and shipped refrigerated. Alternatively, they may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively, the kits may include the active agents premixed and shipped in a vial, tube, or other container. In some embodiments, kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc.

[0184] In some embodiments, may further comprise components needed for inducing uptake of any of the engineered acids or rAAV particles (.e.g., pseudotyped avian rAAV particles) described herein into a cell. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

[0185] In some embodiments, a kit further comprises a set of instructions for carrying out the methods described herein. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of this disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. In some embodiments, written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral, and electronic communication of any form, associated with this disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[0186] In some embodiments, the kits may have a variety of forms, such as a blister pouch, a shrink-wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box, or a bag. In some embodiments, kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. In some embodiments, kits, or any of its components, can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art.

[0187] EXAMPLES

[0188] Example 1: In vitro production of pseudotyped avian rAAV particles

[0189] This Example describes methods for in vitro production of pseudotyped avian rAAV particles using cells lines. FIGs. 2A-2B and 4 show examples of a transfection strategy according to the methods described herein for production of pseudotyped avian rAAV particles in mammalian (e.g., HEK293T) and avian (e.g., QT6) host cells. Suitable host cells are transfected with a plurality of nucleic acid constructs (e.g., see FIG. 1 IB-11G, Tables 1-4 and 6- 7, and SEQ ID NOs: 1, 15, 17, 19, 23, and 32) under conditions described herein to promote production of pseudotyped avian rAAV particles. At a minimum, the plurality of engineered nucleic acids comprises the first engineered nucleic acid and the second engineered nucleic acid, wherein the first engineered nucleic acid comprises a heterologous nucleic flanked by avian AAV ITRs, and wherein the second engineered nucleic acid comprises gene sequences encoding avian AAV rep and AAV capsid proteins. In some embodiments, the constituent engineered nucleic acids of the plurality are contacted with the manufacturing system concurrently. In some embodiments, the constituent engineered nucleic acids of the plurality are contacted with a manufacturing system in a step-wise manner wherein an appropriate time period (e.g., on the order of seconds, tens of seconds, minutes, tens of minutes, hours, etc.) separates each engineered nucleic acid being contacted with the manufacturing system.

[0190] Assembled particles are then recovered, isolated, and, in some instances, purified. Therein, enriched particles are contacted with cell populations to promote transduction and reinfection leading to propagation of said viruses. Example 2: Examples of Vectors for Production of Pseudotyped Avian rAAV Particles

[0191] This Example describes data supporting suitable engineered nucleic acids and transgene expression methods for production of pseudotyped avian rAAV particles.

[0192] In some embodiments, an engineered nucleic acid (e.g., a vector) for pseudotyped avian rAAV particle production comprises one or more of the sequences set forth in Tables 1-4 and 6- 7 (see, e.g., SEQ ID NOs: 1-20 and 23-35). In some embodiments, an engineered nucleic acid (e.g., a vector) for pseudotyped avian rAAV particle production is made by modifying and / or combining any one or more of SEQ ID NOs: 1-35 using known methods. See, e.g., Sambrook et al., Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; (1989), or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989), and DNA Cloning: A Practical Approach, Volumes I and II (ed. D. N. Glover) IREL Press, Oxford, (1985).

[0193] The engineered nucleic acids described in Tables 1-4 and 6-7 were generated and used for production of pseudotyped rAAV particles as described herein. FIGs. 12A-12D show fluorescent microscopy analyses of GFP expression from a pseudotyped avian rAAV. FIG. 12A shows GFP expression in the chorioallantoic membrane (CAM) of an embryonated chicken egg that was inoculated with pseudotyped avian rAAV vectors. The clustered staining the CAM images corresponded to increased expression of the GFP marker relative to controls. FIG. 12B shows GFP expression in chicken embryonic cells (CEC; derived from either brain or body tissues) transduced with pseudotyped avian rAAV particles comprising the indicated capsid proteins made in HEK293T cells. HEK293T cells comprising the corresponding pseudotyped avian rAAV vectors are shown as a control. The results indicated that transgene expression was higher in the CEC cells as compared to HEK293T control cells. FIG. 12C shows GFP expression in CECs transduced with pseudotyped avian rAAV particles. Avian QT6 cells comprising the corresponding pseudotyped avian rAAV vectors are shown as a control. The results indicated that CEC cells exhibited high transgene expression relative to control cells. FIG. 12D shows GFP expression in HEK293T cells contacted with a pseudotyped avian rAAV particle comprising a GFP gene flanked by avian AAV2 ITRs (left panel) and adult mouse brain cells contacted with a pseudotyped avian rAAV particle comprising a GFP gene and a mammalian AAV5 capsid protein (right panel).

[0194] As an additional example, in some embodiments, a vector may be engineered to comprise one or more sequences derived from CELO virus. In some embodiments, the vector comprises sequences from the pBeloBacl l plasmid. CELO virus was purchased from Charles River and virus concentrated overnight by using PEG / NaCl solution (e.g., see step 1 of FIG. 13A). Virus genomic DNA was purified using Sigma REDExtract-N-Amp™ Tissue PCR Kit from virus overnight pellet. The pBeloBacl 1 plasmid from NEB was used to clone the virus genome. BamHI and Hindlll sites were used for cloning. PCR was used to amplify both CELO virus ITR (5’-ITR and 3’-ITR) into the pBeloBacl 1 BamHI and Hindlll sites. Two PacI sites were introduced during the PCR to each ITR. 5 ’-ITR will have PacI at the beginning and 3 ’-ITR will have PacI at the end. Future PacI digestion will release the CELO virus genome from the plasmid. A single Pmel site was introduced between the two ITR to allow their separation. This plasmid was designated as pBeloBacl 1-CELO-ITRs (e.g., see step 2 of FIG. 13A). The pBeloBacl 1-CELO-ITRs was digested with Pmel to open the plasmid, CIP treated to inhibit self-ligation, and Taq treated with dNTPs to inhibit self-ligation. The cut plasmid and CELO genomic DNA (gDNA) were combined with BJ5183 bacteria cells (Agilent) and electroporated. BJ5183 cells facilitated the homologous recombination of the virus genome with pBeloBacl 1-CELO-ITRs plasmid resulting in the cloning of the CELO virus genome after selection using Chloramphenicol LB plates. Cloned CELO genome plasmid was designated as “pBeloBacl 1-CELO-gDNA-wt” (see, e.g., step 3 of FIG. 13A-13B).

[0195] Restriction digestion and whole plasmid sequencing confirmed the pBeloBacl 1-CELO- gDNA-wt (see, e.g., step 4 of FIGs. 13A-13C and Table 9). FIG. 13D shows GFP expression in CECs transduced with pseudotyped avian rAAV particles which were generated using a nucleic acid comprising a GFP transgene operably linked to a CMV promoter flanked by avian AAV ITRs. The pBeloBacl 1-CELO-gDNA-wt nucleic acid was used to provide helper functions. A nucleic acid comprising avian AAV rep and a non-avian AAV cap sequence provided on a pAdCEV vector (provided by Avril Biopharma, Inc.) provided packaging functions. For comparison, avian QT6 cells comprising pseudotyped avian rAAV vectors generated using helper functions of pHelper plasmid and CELO virus gDNA are also shown. The results indicated that CEC cells exhibited high transgene expression when pBeloBacl 1-CELO-gDNA- wt nucleic acid was used to provide helper functions.

[0196] Novel AAV production vectors were presented here that were constructed specifically for use in avian eggs and avian cell line cultures. These vectors utilized the Avian AAV pseudotyping technology, also incorporating CELO virus genome origin of replication allowing amplification of the vectors within avian cells (e.g., avian cell lines and cells in avian eggs). Avian AAV pseudotyping transgene vectors were duplicated by two different mechanisms in the avian cells (e.g., avian cell lines and cells in avian eggs). One duplication was driven by the CELO virus and the second by avian AAV rep protein. This extra replication of the Avian AAV pseudotyping vector in the avian cells (e.g., avian cell lines and cells in avian eggs) yielded higher titers of pseudo-typed virus per cell. In summary, CELO virus can be used for two purposes in avian cell production of AAVs in eggs or in cell culture: (1) providing a safer helper function for production of human AAV particles and (2) providing a novel amplifications function to the replication of AAV producing vectors.

[0197] INCORPORATION BY REFERENCE

[0198] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

[0199] EQUIVALENTS

[0200] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0201] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0202] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0203] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, e.g., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0204] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (e.g., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0205] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0206] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, e.g., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

[0207] NUMERATED EMBODIMENTS

[0208] The following numerated embodiments represent non-limiting aspects of the disclosure:

[0209] 1. A plurality of nucleic acids comprising: i) a first nucleic acid comprising a heterologous nucleic acid flanked by a first and second avian adeno-associated virus (AAV) inverted terminal repeat (ITR), and ii) a second nucleic acid comprising at least one gene sequence encoding an AAV capsid protein and at least one gene sequence encoding an avian AAV rep protein.

[0210] 2. The plurality of nucleic acids of embodiment 1, wherein the heterologous nucleic acid comprises a gene sequence encoding an RNA, wherein the gene sequence is operably linked to at least one regulatory sequence.

[0211] 3. The plurality of nucleic acids of embodiment 2, wherein the at least one regulatory sequence is selected from the group consisting of a promoter, an enhancer, a poly(A) signal, an intron, a Woodchuck Hepatitis Virus Post-Transcriptional Response Element (WPRE), a splicing donor / splicing acceptor element, and a bipartite leader (BPL) sequence comprising a Kozak sequence.

[0212] 4. The plurality of nucleic acids of embodiment 3, wherein the promoter is a CMV promoter.

[0213] 5. The plurality of nucleic acids of embodiment 3 or 4, wherein the enhancer is a CMV enhancer.

[0214] 6. The plurality of nucleic acids of any one of embodiments 3-5, wherein the poly(A) signal is a Proudfoot poly(A) signal.

[0215] 7. The plurality of nucleic acids of any one of embodiments 1-6, wherein the heterologous nucleic acid comprises from 5’ to 3’, in relative order, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, the gene sequence encoding an RNA, and a Proudfoot poly(A) signal.

[0216] 8. The plurality of nucleic acids of any one of embodiments 1-7, wherein the gene sequence encodes a therapeutic RNA or therapeutic protein.

[0217] 9. The plurality of nucleic acids of embodiment 8, wherein the therapeutic protein is therapeutic for a genetic disease, cancer, inflammatory disease or inflammatory condition, autoimmune disease, liver disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or any combination thereof.

[0218] 10. The plurality of nucleic acids of embodiment 8 or 9, wherein the therapeutic RNA is selected from the group consisting of an interfering RNA, exon-skipping RNA, enzymatic RNA, guide RNA, small nuclear RNA, ribosomal RNA, and transfer RNA, wherein the therapeutic protein is selected from the group consisting of enzyme, enzymatic domain, enzyme substrate, hormone, receptor, gene editor, peptibody, growth factor, clotting factor, cytokine, chemokine, ion channel-activating or inhibitory peptide, cell-permeable peptide, thrombolytic, bone morphogenetic protein, Fc-fusion protein, anticoagulant, and antibody or antigen-binding fragment thereof.

[0219] 11. The plurality of nucleic acids of any one of embodiments 1-10, wherein the heterologous nucleic acid comprises a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 25-29.

[0220] 12. The plurality of nucleic acids of any one of embodiments 1-10, wherein the heterologous nucleic acid comprises the sequence of any one of SEQ ID NOs: 25-29.

[0221] 13. The plurality of nucleic acids of any one of embodiments 1-12, wherein the first nucleic acid is a vector comprising from 5’ to 3’, in relative order, a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, the first avian AAV ITR, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, the therapeutic gene sequence, and a Proudfoot poly(A) signal, the second avian AAV ITR, and a bovine growth hormone (bGH) poly (A) signal, wherein the vector further comprises a 5’ chicken embryo lethal orphan (CELO) virus ITR and a 3’ CELO virus ITR.

[0222] 14. The plurality of nucleic acids of any one of embodiments 1-13, wherein the vector comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 23-31. 15. The plurality of nucleic acids of any one of embodiments 1-13, wherein the vector comprises a nucleic acid sequence of any one of SEQ ID NOs: 23-31.

[0223] 16. The plurality of nucleic acids of any one of embodiments 1-15, wherein the at least one AAV capsid protein is selected from the group consisting of a mammalian AAV1, mammalian AAV5, mammalian AAV6, mammalian AAV7 mammalian AAV9, and mammalian AAV8-M3.

[0224] 17. The plurality of nucleic acids of any one of embodiments 1-16, wherein the second engineered nucleic acid is a vector comprising from 5’ to 3’, in relative order, a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, the at least one gene sequence encoding an avian rep protein, the at least one gene sequence encoding an AAV capsid protein, and a bovine growth hormone (bGH) poly (A) signal, wherein the vector further comprises a 5’ CELO virus ITR and a 3’ CELO virus ITR.

[0225] 18. The plurality of nucleic acids of any one of embodiments 1-17, further comprising a helper nucleic acid.

[0226] 19. The plurality of nucleic acids of embodiment 18, wherein the helper nucleic acid comprises at least one of El, E2A, E4, or VA.

[0227] 20. The plurality of nucleic acids of embodiment 18, wherein helper nucleic acid is provided on a third nucleic acid, optionally wherein the third nucleic acid is a vector comprising a sequence that is at least 75% identical to SEQ ID NO: 36.

[0228] 21. The plurality of nucleic acids of any one of embodiments 1-20, wherein the second nucleic acid comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 1-20 or 32-33.

[0229] 22. The plurality of nucleic acids of any one of embodiments 1-20, wherein the second engineered nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-20 or 32-33.

[0230] 23. A manufacturing system comprising the plurality of nucleic acids of any one of embodiments 1-22, wherein the manufacturing system is an avian egg or a host cell. 24. The manufacturing system of embodiment 23, wherein the avian egg is selected from the group consisting of chicken egg, duck egg, goose egg, and quail egg.

[0231] 25. The manufacturing system of embodiment 24, wherein the avian egg is an embryonated egg-

[0232] 26. The manufacturing system of embodiment 23, wherein the host cell is a cell of a cell line.

[0233] 27. The manufacturing system of embodiment 23 or 26, wherein the host cell is an avian cell.

[0234] 28. The manufacturing system of embodiment 27, wherein the avian cell is selected from the group consisting of chicken cell, duck cell, goose cell, and quail cell.

[0235] 29. The manufacturing system of embodiment 28, wherein the chicken cell is a EB14 cell, HD-11 cell, DF-1 cell, DT95 cell, DT40 cell, LMH cell, or PBS-12 SF cell.

[0236] 30. The manufacturing system of embodiment 28, wherein the duck cell is an AGE1.CR cell, AGEl.CR.pIX cell, EB66 cell, or DEF cell.

[0237] 31. The manufacturing system of embodiment 28, wherein the quail cell is a QEF cell, a QM7 cell, a QT-35 cell, or a QT-6 cell.

[0238] 32. The manufacturing system of any one of embodiments 26-31, wherein the host cell is a suspension cell.

[0239] 33. The manufacturing system of embodiment 26, wherein the cell is a HEK293 cell.

[0240] 34. The manufacturing system of any one of embodiments 26-34, wherein the manufacturing system is a cell population comprising a plurality of the host cells. 35. The manufacturing system of any one of embodiments 23-34, further comprising a helper nucleic acid.

[0241] 36. The manufacturing system of any one of embodiments 23-35, wherein the helper nucleic acid is an adenovirus nucleic acid.

[0242] 37. The manufacturing system of embodiment 36, wherein the helper nucleic acid is an avian adenovirus nucleic acid.

[0243] 38. The manufacturing system of embodiment 37, wherein the helper nucleic acid is a CELO helper virus nucleic acid, optionally wherein the CELO helper virus nucleic acid comprises at least 75% identity to SEQ ID NO: 36.

[0244] 39. A method of producing an rAAV particle comprising: i) contacting a manufacturing system with the plurality of nucleic acids of any one of embodiments 1-22, ii) incubating the manufacturing system, and iii) isolating the rAAV particle from the manufacturing system thereby yielding an isolated rAAV particle.

[0245] 40. The method of embodiment 39, wherein manufacturing system is a host cell or an avian egg-

[0246] 41. The method of embodiment 39, wherein the avian egg is a chicken egg, duck egg, quail egg, or goose egg.

[0247] 42. The method of embodiment 40 or 41, wherein the avian egg is an embryonated egg.

[0248] 43. The method of embodiment 40, wherein the host cell is an avian cell

[0249] 44. The method of embodiment 43, wherein the avian cell is selected from the group consisting of chicken cell, duck cell, goose cell, and quail cell. 45. The method of embodiment 44, wherein the chicken cell is a EB14 cell, HD-11 cell, DF- 1 cell, DT95 cell, DT40 cell, LMH cell, or PBS- 12 SF cell.

[0250] 46. The method of embodiment 44, wherein the duck cell is an AGE1.CR cell, AGEl.CR.pIX cell, EB66 cell, or DEF cell.

[0251] 47. The method of embodiment 44, wherein the quail cell is a QEF cell, a QM7 cell, a QT- 35 cell, or a QT-6 cell.

[0252] 48. The method of any one of embodiments 40 or 43-47, wherein the host cell is a suspension cell.

[0253] 49. The method of embodiment 40, wherein the host cell is a HEK293 cell.

[0254] 50. The method of any one of embodiments 40 or 43-49, wherein the manufacturing system is a cell population comprising a plurality of the host cells.

[0255] 51. The method of any one of embodiments 39-50, wherein the manufacturing system further comprises a helper nucleic acid.

[0256] 52. The method of embodiment 51, wherein the helper nucleic acid comprises at least one of E1, E2A, E4, or VA.

[0257] 53. The method of 51, wherein the helper nucleic acid is an adenovirus nucleic acid.

[0258] 54. The method of embodiment 53, wherein the helper nucleic acid is an avian adenovirus nucleic acid.

[0259] 55. The method of embodiment 54, wherein the helper nucleic acid is a CELO helper virus nucleic acid, optionally wherein the CELO helper virus nucleic acid is at least 75% identical to SEQ ID NO: 36.

[0260] 56. The method of any one of embodiments 40-55, wherein contacting comprises inoculating the avian egg.

[0261] I l l 57. The method of embodiment 56, wherein an allantoic cavity, a chorioallantoic membrane, a yolk sac, or an amnion of the avian egg is inoculated.

[0262] 58. The method of embodiment 56 or 57, wherein inoculation comprises manual injection.

[0263] 59. The method of any one of embodiments 40-42 or 51-58, wherein the avian egg is incubated in an automatic egg incubator.

[0264] 60. The method of any one of embodiments 40-42 or 51-59, wherein the avian egg is incubated after inoculation for a period of at least 15 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 80 hours, or at least 90 hours.

[0265] 61. The method of any one of embodiments 39-60, wherein the isolated rAAV particle is substantially free of avian viruses.

[0266] 62. The method of any one of embodiments 40-42 or 51-61, wherein the isolated rAAV particle is isolated from the allantoic fluid.

[0267] 63. The method of any one of embodiments 40-42 or 51-62, wherein the isolated rAAV particle is isolated using a manual pipette, a manual syringe, a machine-controlled pipette, or a machine-controlled syringe.

[0268] 64. The method of any one of embodiments 39-63, further comprising a purification step, wherein the isolated rAAV particle is subjected to iodixanol gradient ultracentrifugation and / or contacted with an affinity resin, thereby producing a purified rAAV particle.

[0269] 65. The method of any one of embodiments 39-64, wherein the isolated rAAV particle or purified rAAV particle is at a titer of at least about 1 x 108, at least about 5 x 108, at least about 1 x 109, at least about 5 x 109, at least about 1 x 1010, at least about 5 x 1010, at least about 1 x 1011, at least 2 x 1011, or at least 5 x 1011vector genomes (vg) / ml. 66. An rAAV particle produced by the method of any one of embodiments 39-65, wherein the rAAV particle comprises at least one AAV capsid protein.

[0270] 67. An rAAV particle comprising:

[0271] (i) the first nucleic acid of any one of embodiments 1-16; and

[0272] (ii) at least one AAV capsid protein.

[0273] 68. The rAAV particle of embodiment 66 or 67, wherein the at least one AAV capsid protein comprises a mammalian AAV1, mammalian AAV5, mammalian AAV6, mammalian AAV7 mammalian AAV9, and mammalian AAV8-M3.

[0274] 69. A method of producing a pharmaceutical composition, comprising:

[0275] (i) performing the method of any one of embodiments 39-66 and combining the isolated and / or purified rAAV particle with a pharmaceutically acceptable ingredient; or

[0276] (ii) combining the rAAV particle of any one of embodiments 66-68 with a pharmaceutically acceptable ingredient.

[0277] 70. A pharmaceutical composition produced by the method of embodiment 69.

[0278] 71. A pharmaceutical composition comprising the rAAV particle of any one of embodiments 66-68 and a pharmaceutically acceptable ingredient.

[0279] 72. A method of treatment comprising administering to a subject in need thereof:

[0280] (i) the rAAV particle of any one of embodiments 66-68;

[0281] (ii) the pharmaceutical composition produced by the method of 69; or

[0282] (iii) the pharmaceutical composition of embodiment 70 or 71.

[0283] 73. The method of embodiment 72, wherein the subject is a human.

[0284] 74. The method of embodiment 72 or 73, wherein the subject has, is suspected of having, or is at risk of developing a genetic disease, cancer, inflammatory disease or inflammatory condition, autoimmune disease, liver disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or any combination thereof.

[0285] 75. The method of any one of embodiments 72-74, wherein administration of the rAAV particle results in lowered AAV-induced toxicity in the subject, optionally wherein the subject has, is suspected of having, or is at risk of developing Aromatic L- Amino Acid Decarboxylase Deficiency, Lever Congenital Amaurosis Disease, Duchenne Muscular Dystrophy (DMD), Hemophilia, Spinal Muscular Atrophy (SMA), Friedreich’s Ataxia, Pompe Disease, Alpha- 1 Antitrypsin Deficiency, X-linked Severe Combinate Immunodeficiency Disease (SCID), or Cystic Fibrosis.

[0286] 76. A vector comprising from 5’ to 3’, in relative order, a gene sequence encoding an avian AAV rep protein and a gene sequence encoding an AAV capsid protein, wherein the vector further comprises a 5’ CELO virus ITR and a 3’ CELO virus ITR.

[0287] 77. The vector of embodiment 76, wherein the vector comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NOs: 34-45.

[0288] 78. The vector of embodiment 77, wherein the vector comprises a nucleic acid sequence of any one of SEQ ID NOs: 34-35.

Claims

CLAIMSWhat is claimed:

1. A plurality of nucleic acids comprising: i) a first nucleic acid comprising a heterologous nucleic acid flanked by a first and second avian adeno-associated virus (AAV) inverted terminal repeat (ITR), and ii) a second nucleic acid comprising at least one gene sequence encoding an AAV capsid protein and at least one gene sequence encoding an avian AAV rep protein.

2. The plurality of nucleic acids of claim 1, wherein the heterologous nucleic acid comprises a gene sequence encoding an RNA, wherein the gene sequence is operably linked to at least one regulatory sequence.

3. The plurality of nucleic acids of claim 2, wherein the at least one regulatory sequence is selected from the group consisting of a promoter, an enhancer, a poly(A) signal, an intron, a Woodchuck Hepatitis Virus Post-Transcriptional Response Element (WPRE), a splicing donor / splicing acceptor element, and a bipartite leader (BPL) sequence comprising a Kozak sequence.

4. The plurality of nucleic acids of claim 3, wherein the promoter is a CMV promoter.

5. The plurality of nucleic acids of claim 3 or 4, wherein the enhancer is a CMV enhancer.

6. The plurality of nucleic acids of any one of claims 3-5, wherein the poly(A) signal is aProudfoot poly (A) signal.

7. The plurality of nucleic acids of any one of claims 1-6, wherein the heterologous nucleic acid comprises from 5’ to 3’, in relative order, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, the gene sequence encoding an RNA, and a Proudfoot poly(A) signal.

8. The plurality of nucleic acids of any one of claims 1-7, wherein the gene sequence encodes a therapeutic RNA or therapeutic protein.

9. The plurality of nucleic acids of claim 8, wherein the therapeutic protein is therapeutic for a genetic disease, cancer, inflammatory disease or inflammatory condition, autoimmune disease, liver disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or any combination thereof.

10. The plurality of nucleic acids of claim 8 or 9, wherein the therapeutic RNA is selected from the group consisting of an interfering RNA, exon- skipping RNA, enzymatic RNA, guide RNA, small nuclear RNA, ribosomal RNA, and transfer RNA, wherein the therapeutic protein is selected from the group consisting of enzyme, enzymatic domain, enzyme substrate, hormone, receptor, gene editor, peptibody, growth factor, clotting factor, cytokine, chemokine, ion channel-activating or inhibitory peptide, cell-permeable peptide, thrombolytic, bone morphogenetic protein, Fc-fusion protein, anticoagulant, and antibody or antigen-binding fragment thereof.

11. The plurality of nucleic acids of any one of claims 1-10, wherein the heterologous nucleic acid comprises a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 25-29.

12. The plurality of nucleic acids of any one of claims 1-10, wherein the heterologous nucleic acid comprises the sequence of any one of SEQ ID NOs: 25-29.

13. The plurality of nucleic acids of any one of claims 1-12, wherein the first nucleic acid is a vector comprising from 5’ to 3’, in relative order, a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, the first avian AAV ITR, a CMV enhancer, a CMV promoter, a splicing donor / splicing acceptor element, the therapeutic gene sequence, and a Proudfoot poly(A) signal, the second avian AAV ITR, and a bovine growth hormone (bGH) poly (A) signal, wherein the vector further comprises a 5’ chicken embryo lethal orphan (CELO) virus ITR and a 3’ CELO virus ITR.

14. The plurality of nucleic acids of any one of claims 1-13, wherein the vector comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 23-31.

15. The plurality of nucleic acids of any one of claims 1-13, wherein the vector comprises a nucleic acid sequence of any one of SEQ ID NOs: 23-31.

16. The plurality of nucleic acids of any one of claims 1-15, wherein the at least one AAV capsid protein is selected from the group consisting of a mammalian AAV1, mammalian AAV5, mammalian AAV6, mammalian AAV7 mammalian AAV9, and mammalian AAV8-M3.

17. The plurality of nucleic acids of any one of claims 1-16, wherein the second engineered nucleic acid is a vector comprising from 5’ to 3’, in relative order, a major late promoter (MLP), a BPL sequence comprising a Kozak sequence, the at least one gene sequence encoding an avian rep protein, the at least one gene sequence encoding an AAV capsid protein, and a bovine growth hormone (bGH) poly (A) signal, wherein the vector further comprises a 5’ CELO virus ITR and a 3’ CELO virus ITR.

18. The plurality of nucleic acids of any one of claims 1-17, further comprising a helper nucleic acid.

19. The plurality of nucleic acids of claim 18, wherein the helper nucleic acid comprises at least one of El, E2A, E4, or VA.

20. The plurality of nucleic acids of claim 18, wherein helper nucleic acid is provided on a third nucleic acid, optionally wherein the third nucleic acid is a vector comprising a sequence that is at least 75% identical to SEQ ID NO: 36.

21. The plurality of nucleic acids of any one of claims 1-20, wherein the second nucleic acid comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 1-20 or 32-33.

22. The plurality of nucleic acids of any one of claims 1-20, wherein the second engineered nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-20 or 32-33.

23. A manufacturing system comprising the plurality of nucleic acids of any one of claims 1- 22, wherein the manufacturing system is an avian egg or a host cell.

24. The manufacturing system of claim 23, wherein the avian egg is selected from the group consisting of chicken egg, duck egg, goose egg, and quail egg.

25. The manufacturing system of claim 24, wherein the avian egg is an embryonated egg.

26. The manufacturing system of claim 23, wherein the host cell is a cell of a cell line.

27. The manufacturing system of claim 23 or 26, wherein the host cell is an avian cell.

28. The manufacturing system of claim 27, wherein the avian cell is selected from the group consisting of chicken cell, duck cell, goose cell, and quail cell.

29. The manufacturing system of claim 28, wherein the chicken cell is a EB14 cell, HD-11 cell, DF-1 cell, DT95 cell, DT40 cell, LMH cell, or PBS- 12 SF cell.

30. The manufacturing system of claim 28, wherein the duck cell is an AGE1.CR cell, AGEl.CR.pIX cell, EB66 cell, or DEF cell.

31. The manufacturing system of claim 28, wherein the quail cell is a QEF cell, a QM7 cell, a QT-35 cell, or a QT-6 cell.

32. The manufacturing system of any one of claims 26-31, wherein the host cell is a suspension cell.

33. The manufacturing system of claim 26, wherein the cell is a HEK293 cell.

34. The manufacturing system of any one of claims 26-34, wherein the manufacturing system is a cell population comprising a plurality of the host cells.

35. The manufacturing system of any one of claims 23-34, further comprising a helper nucleic acid.

36. The manufacturing system of any one of claims 23-35, wherein the helper nucleic acid is an adenovirus nucleic acid.

37. The manufacturing system of claim 36, wherein the helper nucleic acid is an avian adenovirus nucleic acid.

38. The manufacturing system of claim 37, wherein the helper nucleic acid is a CELO helper virus nucleic acid, optionally wherein the CELO helper virus nucleic acid comprises at least 75% identity to SEQ ID NO: 36.

39. A method of producing an rAAV particle comprising: i) contacting a manufacturing system with the plurality of nucleic acids of any one of claims 1-22, ii) incubating the manufacturing system, and iii) isolating the rAAV particle from the manufacturing system thereby yielding an isolated rAAV particle.

40. The method of claim 39, wherein manufacturing system is a host cell or an avian egg.

41. The method of claim 39, wherein the avian egg is a chicken egg, duck egg, quail egg, or goose egg.

42. The method of claim 40 or 41, wherein the avian egg is an embryonated egg.

43. The method of claim 40, wherein the host cell is an avian cell44. The method of claim 43, wherein the avian cell is selected from the group consisting of chicken cell, duck cell, goose cell, and quail cell.

45. The method of claim 44, wherein the chicken cell is a EB14 cell, HD-11 cell, DF-1 cell, DT95 cell, DT40 cell, LMH cell, or PBS- 12 SF cell.

46. The method of claim 44, wherein the duck cell is an AGE1.CR cell, AGEl.CR.pIX cell, EB66 cell, or DEF cell.

47. The method of claim 44, wherein the quail cell is a QEF cell, a QM7 cell, a QT-35 cell, or a QT-6 cell.

48. The method of any one of claims 40 or 43-47, wherein the host cell is a suspension cell.

49. The method of claim 40, wherein the host cell is a HEK293 cell.

50. The method of any one of claims 40 or 43-49, wherein the manufacturing system is a cell population comprising a plurality of the host cells.

51. The method of any one of claims 39-50, wherein the manufacturing system further comprises a helper nucleic acid.

52. The method of claim 51, wherein the helper nucleic acid comprises at least one of El, E2A, E4, or VA.

53. The method of 51, wherein the helper nucleic acid is an adenovirus nucleic acid.

54. The method of claim 53, wherein the helper nucleic acid is an avian adenovirus nucleic acid.

55. The method of claim 54, wherein the helper nucleic acid is a CELO helper virus nucleic acid, optionally wherein the CELO helper virus nucleic acid is at least 75% identical to SEQ ID NO: 36.

56. The method of any one of claims 40-55, wherein contacting comprises inoculating the avian egg.

57. The method of claim 56, wherein an allantoic cavity, a chorioallantoic membrane, a yolk sac, or an amnion of the avian egg is inoculated.

58. The method of claim 56 or 57, wherein inoculation comprises manual injection.

59. The method of any one of claims 40-42 or 51-58, wherein the avian egg is incubated in an automatic egg incubator.

60. The method of any one of claims 40-42 or 51-59, wherein the avian egg is incubated after inoculation for a period of at least 15 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 80 hours, or at least 90 hours.

61. The method of any one of claims 39-60, wherein the isolated rAAV particle is substantially free of avian viruses.

62. The method of any one of claims 40-42 or 51-61, wherein the isolated rAAV particle is isolated from the allantoic fluid.

63. The method of any one of claims 40-42 or 51-62, wherein the isolated rAAV particle is isolated using a manual pipette, a manual syringe, a machine-controlled pipette, or a machine- controlled syringe.

64. The method of any one of claims 39-63, further comprising a purification step, wherein the isolated rAAV particle is subjected to iodixanol gradient ultracentrifugation and / or contacted with an affinity resin, thereby producing a purified rAAV particle.

65. The method of any one of claims 39-64, wherein the isolated rAAV particle or purified rAAV particle is at a titer of at least about 1 x 108, at least about 5 x 108, at least about 1 x 109, at least about 5 x 109, at least about 1 x 1010, at least about 5 x 1010, at least about 1 x 1011, at least 2 x 1011, or at least 5 x 1011vector genomes (vg) / ml.

66. An rAAV particle produced by the method of any one of claims 39-65, wherein the rAAV particle comprises at least one AAV capsid protein.

67. An rAAV particle comprising:(i) the first nucleic acid of any one of claims 1-16; and(ii) at least one AAV capsid protein.

68. The rAAV particle of claim 66 or 67, wherein the at least one AAV capsid protein comprises a mammalian AAV1, mammalian AAV5, mammalian AAV6, mammalian AAV7 mammalian AAV9, and mammalian AAV8-M3.

69. A method of producing a pharmaceutical composition, comprising:(i) performing the method of any one of claims 39-66 and combining the isolated and / or purified rAAV particle with a pharmaceutically acceptable ingredient; or(ii) combining the rAAV particle of any one of claims 66-68 with a pharmaceutically acceptable ingredient.

70. A pharmaceutical composition produced by the method of claim 69.

71. A pharmaceutical composition comprising the rAAV particle of any one of claims 66-68 and a pharmaceutically acceptable ingredient.

72. A method of treatment comprising administering to a subject in need thereof:(i) the rAAV particle of any one of claims 66-68;(ii) the pharmaceutical composition produced by the method of 69; or(iii) the pharmaceutical composition of claim 70 or 71.

73. The method of claim 72, wherein the subject is a human.

74. The method of claim 72 or 73, wherein the subject has, is suspected of having, or is at risk of developing a genetic disease, cancer, inflammatory disease or inflammatory condition, autoimmune disease, liver disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or any combination thereof.

75. The method of any one of claims 72-74, wherein administration of the rAAV particle results in lowered AAV-induced toxicity in the subject, optionally wherein the subject has, issuspected of having, or is at risk of developing Aromatic L- Amino Acid Decarboxylase Deficiency, Lever Congenital Amaurosis Disease, Duchenne Muscular Dystrophy (DMD), Hemophilia, Spinal Muscular Atrophy (SMA), Friedreich’s Ataxia, Pompe Disease, Alpha- 1 Antitrypsin Deficiency, X-linked Severe Combinate Immunodeficiency Disease (SCID), or Cystic Fibrosis.

76. A vector comprising from 5’ to 3’, in relative order, a gene sequence encoding an avian AAV rep protein and a gene sequence encoding an AAV capsid protein, wherein the vector further comprises a 5’ CELO virus ITR and a 3’ CELO virus ITR.

77. The vector of claim 76, wherein the vector comprises a nucleic acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NOs: 34-45.

78. The vector of claim 77, wherein the vector comprises a nucleic acid sequence of any one of SEQ ID NOs: 34-35.

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