AAV transfer plasmid

Abstract

Disclosed herein are nucleic acid stuffer sequences with one or more of the following characteristics: no CpG islands and no more than four consecutive nucleobases of the same identity, and no open reading frames greater than 20 amino acids in length. In some embodiments, the nucleic acid stuffer sequence comprises a GC content of about 40% to about 50%. In some embodiments, the nucleic acid stuffer sequence does not comprise a restriction enzyme cleavage site. In some embodiments, the nucleic acid stuffer sequence has a length of about 100 nucleobases to about 5000 nucleobases.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 226,410, filed July 28, 2021, which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of sequence listing This application has been filed with an electronic format Sequence Listing, which was created on July 25, 2022, is provided as a file entitled 58774-726_601_SL.XML, and is 152 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. Summary of the Invention

[0003] In some aspects, the nucleic acid stuffer sequence comprises one or more of the following features: no CpG islands and no more than four consecutive nucleobases of the same identity, and no open reading frame greater than 20 amino acids in length. In some embodiments, the nucleic acid stuffer sequence does not have a CPG island. In some embodiments, the nucleic acid stuffer sequence has no more than four consecutive nucleobases of the same identity. In some embodiments, the nucleic acid stuffer sequence comprises a GC content of about 40% to about 50%. In some embodiments, the nucleic acid stuffer sequence does not comprise a restriction enzyme cleavage site. In some embodiments, the nucleic acid stuffer sequence has a length of about 100 nucleobases to about 5000 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 2200 nucleobases to about 2300 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 3000 nucleobases to about 3100 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 300 nucleobases to about 400 nucleobases.

[0004] In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous bases of SEQ ID NO:7, 8, or 11. In certain aspects, disclosed herein are nucleic acid stuffer sequences that comprise a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous bases of SEQ ID NO:7. In certain aspects, disclosed herein are nucleic acid stuffer sequences comprising a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous bases of SEQ ID NO: 8. In certain aspects, disclosed herein are nucleic acid stuffer sequences comprising a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous bases of SEQ ID NO: 11. In some embodiments, the nucleic acid stuffer sequence does not include a CPG island. In some embodiments, the nucleic acid stuffer sequence comprises no more than four consecutive nucleobases of the same identity. In some embodiments, the nucleic acid stuffer sequence comprises a GC content of about 40% to about 50%. In some embodiments, the nucleic acid stuffer sequence does not comprise a restriction enzyme cleavage site. In some embodiments, the nucleic acid stuffer sequence has a length of about 100 to about 5000 nucleobases. In some embodiments, disclosed herein is an adeno-associated virus (AAV) plasmid comprising a nucleic acid stuffer sequence disclosed herein. In some embodiments, the AAV plasmid further comprises an expression cassette comprising a heterologous sequence located between two inverted terminal repeat (ITR) sequences.In some embodiments, the AAV plasmid further comprises a backbone having a length of about 2000 to about 8000 nucleobases. In some embodiments, the AAV plasmid further comprises a backbone having a length of about 5500 to about 6000 nucleobases. In some embodiments, the expression cassette has a length of about 3000 to about 6000 nucleobases. In some embodiments, the expression cassette has a length of about 4000 nucleobases to about 5000 nucleobases. In some embodiments, the heterologous sequence encodes a therapeutic peptide. In some embodiments, the therapeutic peptide is selected from the group consisting of GUCY2D, MYO7A, RS1, CNBG3, ADAMTS10, ABCA4, and frataxin. In some embodiments, the AAV plasmid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene comprises a kanamycin resistance gene. In some embodiments, the AAV plasmid does not comprise an antibiotic resistance gene. In some embodiments, the AAV plasmid does not comprise an ampicillin antibiotic resistance gene. In some embodiments, the ITRs are derived from AAV serotype 1, AAV serotype 2, AAV serotype 3, AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, or AAV449.5(e531d). In some embodiments, the AAV plasmid further comprises a promoter. In some embodiments, the AAV plasmid further comprises a splice donor / splice acceptor sequence. In some embodiments, the AAV plasmid further comprises a WPRE sequence. In some embodiments, the nucleic acid stuffer sequence is located outside the expression cassette. In some embodiments, the AAV plasmid further comprises an origin of replication. In some embodiments, the nucleic acid stuffer sequence is located 3' to the origin of replication. In some embodiments, the nucleic acid stuffer sequence is located between the origin of replication and the ITR. In some embodiments, the nucleic acid stuffer sequence is located such that the ITR is about 1000 to about 4000 nucleobases away from the origin of replication. In some embodiments, the nucleic acid stuffer sequence comprises a first stuffer sequence and a second stuffer sequence.In some embodiments, the first stuffer sequence has a length of about 3000 to about 3500 nucleobases. In some embodiments, the first stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous bases of SEQ ID NO: 8. In some embodiments, the second stuffer sequence has a length of about 100 to about 500 nucleobases. In some embodiments, the second stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous bases of SEQ ID NO: 11. In some embodiments, the nucleic acid stuffer sequence is located within an expression cassette. In some embodiments, the nucleic acid stuffer sequence has a length of about 2000 to about 3000 nucleic acid bases. In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 consecutive bases of SEQ ID NO:7. In some embodiments, the presence of the nucleic acid stuffer sequence reduces mutations in one or both ITRs compared to an AAV plasmid that does not contain a nucleic acid stuffer sequence. In some embodiments, the AAV plasmid has no more than one origin of replication. In some embodiments, the AAV plasmid does not have an m13 origin of replication. In some embodiments, the AAV plasmid does not contain a polyG / C sequence. In some embodiments, the AAV plasmid does not contain a sequence with more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive guanine bases.

[0005] In some embodiments, compositions are disclosed herein that include an AAV plasmid disclosed herein and a packaging plasmid that includes a viral replication (rep) gene and / or a viral capsid (cap) gene. In some embodiments, the packaging plasmid includes the rep gene. In some embodiments, the rep gene encodes rep78, rep68, rep52, and rep40. In some embodiments, the packaging plasmid includes the cap gene. In some embodiments, the cap gene encodes vpl, vp2, and vp3. In some embodiments, the cap gene comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31. In some embodiments, the composition includes a helper plasmid.

[0006] In some embodiments, the present disclosure provides a composition comprising the AAV plasmid described herein and a helper plasmid.In some embodiments, the helper plasmid comprises E1a gene, E1b gene, E4 gene, E2a, e3 gene, E5 gene, Fiber gene, or VA gene, or a combination thereof.In some embodiments, the helper plasmid comprises a mutated Fiber gene.In some embodiments, the helper plasmid comprises a mutated Fiber gene.In some embodiments, the helper plasmid does not comprise a Fiber gene.

[0007] Disclosed herein, in some embodiments, is a cell comprising an AAV plasmid disclosed herein or a composition disclosed herein.

[0008] In some embodiments, disclosed herein are AAV particles comprising a nucleic acid and a capsid, and the AAV particles are produced by the AAV plasmids disclosed herein, the compositions disclosed herein, or the cells disclosed herein. In some embodiments, described herein are pharmaceutical compositions comprising the AAV particles disclosed herein and a pharma- ceutically acceptable carrier, buffer, diluent, or excipient, or any combination thereof.

[0009] In some embodiments, disclosed herein is a method for transducing a cell, comprising administering to the cell an AAV vector disclosed herein, a composition disclosed herein, an AAV particle disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the cell is a photoreceptor cell. In some embodiments, the cell is a retinal pigment epithelium (RPE) cell. In some embodiments, the cell is a retinal ganglion cell.

[0010] In some embodiments, disclosed herein are methods for treating eye diseases or conditions in a mammal, comprising administering to the mammal an AAV particle as disclosed herein or a pharmaceutical composition as disclosed herein. In some embodiments, the disease or condition comprises retinitis pigmentosa, Leber's congenital amaurosis (e.g., LCA10), age-related macular degeneration (AMD), wet AMD, dry AMD, uveitis, Best's disease, Stargardt's disease, Usher syndrome, geographic atrophy, diabetic retinopathy, retinopathy, achromatosis, choroideremia, Bardet-Beer's syndrome, or glycogen storage disease (ocular condition). In some embodiments, administration is to one or both eyes of the mammal. In some embodiments, the AAV particle is administered intravitreally or subretinally.

[0011] In some embodiments, described herein are nucleic acid stuffer sequences disclosed herein, AAV plasmids disclosed herein, compositions disclosed herein, cells disclosed herein, AAV particles disclosed herein, or pharmaceutical compositions disclosed herein for use in treating ocular diseases or conditions. In some embodiments, described herein are uses of nucleic acid stuffer sequences disclosed herein, AAV plasmids disclosed herein, compositions disclosed herein, cells disclosed herein, AAV particles disclosed herein, or pharmaceutical compositions disclosed herein in the manufacture of medicaments for use in treating ocular diseases or conditions. [Brief description of the drawings]

[0012] The novel features of the invention are set forth with particularity in the appended claims. To better understand the features and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings in which:

[0013] [Figure 1] Figure 1A shows a representative example of an AAV backbone plasmid digested with SmaI. Figure 1B shows an AAV backbone plasmid grown in ampicillin-resistant cells and digested with SmaI. Figure 1C shows an AAV backbone plasmid grown in kanamycin-resistant cells and digested with SmaI. [Figure 2A] The polyG / C sequences in the AAV plasmid backbone are shown. [Figure 2B] The AAV backbone plasmid was digested with SmaI to remove the polyG / C sequence. [Diagram 3] FIG. 1 is a schematic showing different processes that can lead to errors in AAV packaging. [Figure 4A] The AAV plasmid backbone is shown with a stuffer sequence located 3' to the origin. [Figure 4B] Shown is the AAV backbone plasmid of FIG. 4A digested with SmaI. [Figure 5A] The AAV plasmid backbone is shown with a stuffer sequence located 5' of the origin. [Figure 5B] Shown is the AAV backbone plasmid of FIG. 5A digested with SmaI. [Figure 6] The AAV plasmid backbone is shown with a stuffer sequence located 5' of the origin and a polyG / C sequence digested with SmaI. [Figure 7] ITR stability is compared between AAV backbone plasmids propagated in ampicillin-resistant or kanamycin-resistant cells. [Figure 8A] A schematic diagram of the pTR-X002-3pSR transfer plasmid used is shown. [Figure 8B] 1 shows the process of constructing pTR-X002-3pSR transfer plasmid. [Figure 8C] Schematic diagram of the Rep / Cap plasmid. [Figure 8D] 1 shows a schematic diagram of the helper plasmid. [Figure 9] 9A-9C show schematic diagrams of six cassettes from exemplary vectors of the present disclosure. Exemplary vectors include pTR-X001-3p (FIG. 9A) having a length of 4534 bp from the 5' end of the first ITR to the 3' end of the second ITR, pTR-X001-5p (FIG. 9B) having a length of 4528 bp from the 5' end of the first ITR to the 3' end of the second ITR, and pTR-X002-3p (FIG. 9C) having a length of 4549 bp from the 5' end of the first ITR to the 3' end of the second ITR. pTR-GRKl-hRS1syn was packaged into AAV5 and AAV.SPR. pTR-CBA-hRS1syn vector plasmid (pTR-UFl1 backbone) was packaged into AAV5 as a control. [Figure 10]Figure 1 shows restoration of retinal structure in RS1KO mice treated with rAAV.SPR containing a stuffed cassette. Quantification of retinoschisis scores in RS1KO mice treated with vehicle or rAAV.SPR vectors containing the following cassettes: X001, X001-3p, X001-5p, or X002-3p. All vectors improved retinoschisis scores at both time points, except for X001-5p at 1 month post-injection. Nominal descriptive statistical significance was determined by two-way ANOVA with Tukey's post-hoc test for treated eyes. [Figure 11A] 11A-11B show restoration of retinal function in RS1KO mice treated with rAAV.SPR containing a stuffed cassette. Mean maximum scotopic (left) and photopic (right) b-wave amplitudes in RS1KO mice were measured 1 and 2 months after subretinal injection in one eye with either vehicle or rAAV.SPR containing the following cassettes: X001, X001-3p, X001-5p, or X002-3p. Vectors were administered at either 1×108 vg (FIG. 11A) or 5×108 vg (FIG. 11B). Retinal function in all vector-treated eyes was improved over untreated control eyes. Nominal descriptive statistical significance was determined by two-way ANOVA with Tukey's post-hoc test for each individual data set. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 11B]11A-11B show restoration of retinal function in RS1KO mice treated with rAAV.SPR containing a stuffed cassette. Mean maximum scotopic (left) and photopic (right) b-wave amplitudes in RS1KO mice were measured 1 and 2 months after subretinal injection in one eye with either vehicle or rAAV.SPR containing the following cassettes: X001, X001-3p, X001-5p, or X002-3p. Vectors were administered at either 1×108 vg (FIG. 11A) or 5×108 vg (FIG. 11B). Retinal function in all vector-treated eyes was improved over untreated control eyes. Nominal descriptive statistical significance was determined by two-way ANOVA with Tukey's post-hoc test for each individual data set. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 12A] Figures 12A-B show RS1 expression in the retinas of RS1KO mice treated with rAAV.SPR-X002-3p. Representative retinal cross sections from RS1KO mice (top) treated with either vehicle or rAAV.SPR containing the X002-3p cassette in one eye only. Vector was administered at either 1 x 108 vg (Figure 12A) or 5 x 108 vg (Figure 12B). Contralateral untreated eyes are shown in the bottom row. All retinas were stained with an antibody raised against RS1 (red) and counterstained with DAPI. [Figure 12B] Figures 12A-B show RS1 expression in the retinas of RS1KO mice treated with rAAV.SPR-X002-3p. Representative retinal cross sections from RS1KO mice (top) treated with either vehicle or rAAV.SPR containing the X002-3p cassette alone in one eye. Vector was administered at either 1 x 108 vg (Figure 12AA) or 5 x 108 vg (Figure 12B). Contralateral untreated eyes are shown in the bottom row. All retinas were stained with an antibody raised against RS1 (red) and counterstained with DAPI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] I. Composition of Matter Recombinant adeno-associated virus (rAAV) vectors have been successfully used for in vivo gene transfer in many preclinical animal models of human disease, as well as for long-term expression of a wide variety of therapeutic agents. AAV vectors have also provided long-term clinical benefits in humans when targeted to immune-privileged sites, such as ocular delivery for Leber's congenital amaurosis. The advantage of this vector is its relatively low immune profile, inducing only limited inflammatory responses, and in some cases even inducing immune tolerance to the transgene product.

[0015] Adeno-associated virus (AAV) is used in ocular gene therapy due to its efficiency, persistence and low immunogenicity. Aspects of the present disclosure relate to recombinant adeno-associated virus (rAAV) particles or preparations of such particles for delivering one or more nucleic acid vectors containing a protein or polypeptide of interest to various tissues, organs, and / or cells.

[0016] Described herein are improved systems for the production and packaging of AAV particles. In some embodiments, the systems and methods described herein improve the efficiency of packaging AAV particles, the accuracy of replicating AAV particles, or a combination thereof.

[0017] A. Stuffer array In some embodiments, nucleic acid stuffer sequences are described herein. As described herein, a "stuffer sequence", interchangeably referred to as a "nucleic acid stuffer sequence", can be a nucleic acid sequence that resizes or adjusts the length of an AAV viral genome sequence to a normal or approximate length. In some embodiments, the nucleic acid stuffer sequence does not contain a CpG island, the nucleic acid stuffer sequence does not contain more than four consecutive nucleic acid bases of the same identity, the nucleic acid stuffer sequence contains about 40% to about 50% GC content, the nucleic acid stuffer sequence does not contain a restriction enzyme cleavage site, or the nucleic acid stuffer sequence does not encode an open reading frame (ORF) larger than 20 amino acids, or a combination thereof. In some embodiments, the nucleic acid stuffer sequence does not contain a CpG island, the nucleic acid stuffer sequence does not contain more than four consecutive nucleobases of the same identity, the nucleic acid stuffer sequence comprises a GC content of about 40% to about 50%, the nucleic acid stuffer sequence does not encode an open reading frame (ORF) larger than 20 amino acids, and the nucleic acid stuffer sequence does not contain a restriction enzyme cleavage site.

[0018] In some embodiments, the nucleic acid stuffer does not include a CpG island. In some embodiments, the CpG island (CGI) comprises a number of CpG dinucleotide repeats. In some embodiments, the CpG island comprises a region at least 100 base pairs in length with a GC content of greater than 50% GC. In some embodiments, the CpG island comprises a region at least 100, 200, 300, 400, 500 or more base pairs in length. In some embodiments, the CpG island comprises a region with a GC content of at least 50%, 60%, 70%, 80%, 90% or greater than 90%.

[0019] In some embodiments, the nucleic acid stuffer sequence does not contain more than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleobases of the same identity. In some embodiments, the nucleic acid stuffer sequence does not contain more than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive adenosines. In some embodiments, the nucleic acid stuffer sequence does not contain more than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive cytosines. In some embodiments, the nucleic acid stuffer sequence does not contain more than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive guanines. In some embodiments, the nucleic acid stuffer sequence does not contain more than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive thymines.

[0020] In some embodiments, the nucleic acid stuffer sequence comprises about 30% to about 60% GC content. In some embodiments, the nucleic acid stuffer sequence comprises about 40% to about 50% GC content. In some embodiments, the nucleic acid stuffer sequence comprises about 45% GC content. In some embodiments, the nucleic acid stuffer sequence comprises about 0%-10% GC content, about 5%-15% GC content, about 10%-20% GC content, about 15%-20% GC content, about 25%-35% GC content, about 30%-40% GC content, about 35%-45% GC content, about 40%-50% GC content, about 45%-55% GC content, about 50%-60% GC content, about 55%-65% GC content, about 60%-70% GC content, about 65%-75% GC content, about 70%-80% GC content, about 75%-85% GC content, about 80%-90% GC content, about 85%-95% GC content, or about 0.5%-100% GC content.

[0021] In some embodiments, the nucleic acid stuffer sequence does not include a restriction enzyme cleavage site. In some embodiments, the restriction enzyme cleavage sites are AatII, AbaSI, AccI, Acc65I, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI §, AgeI-HF®, AhdI, AleI-v2, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI §, ApoI-HF, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI §, BamHI-HF®, BanI, BanII, BbsI §, BbsI-HF®, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI §, BclI-HF, BcoDI, BfaI, BfuAI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI §, BmtI-HF (registered trademark), BpmI, BpuEI, Bpu10I, BsaAI, BsaBI, BsaHI, BsaI-HF (registered trademark) v2, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI §, BsiWI-HF (registered trademark), BslI, BsmAI, BsmBI-v2, BsmFI, BsmI, BsoBI, BspCNI, BspDI, BspEI, BspHI, Bsp1286I, BspMI, BspQI, BsrBI, BsrDI, BsrFI-v2, BsrGI §, BsrGI-HF (registered trademark), BsrI, BssHII, BssSI-v2, BstAPI, BstBI, BstEII §, BstEII-HF (registered trademark), BstNI, BstUI, BstXI, BstYI, BstZ17I-HF (registered trademark), Bsu36I, BtgI, BtgZI, BtsCI, BtsIMutI, BtsI-v2, Cac8I, ClaI, CspCI, CviAI I, CviKI-1, CviQI, DdeI, DpnI, DpnII, DraI, DraIII-HF (registered trademark), DrdI, EaeI, EagI-HF (registered trademark), EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI §, EcoRI-HF(registered trademark), EcoRV§、EcoRV-HF(EcoRV-HF), Esp3I, FatI, FauI, Fnu4HI, FokI, FseI, FspEI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII §, HindIII-HF(free solvent), HinfI, HinP1I, HpaI, HpaII, HphI, HpyAV, HpyCH4III, HpyCH 4IV, HpyCH4V, Hpy188I, Hpy99I, Hpy166II, Hpy188III, I-CeuI, I-SceI, CasI, KpnI §、KpnI-HF(free range)、LpnPI、MboI、MboII、MfeI §、MfeI-HF(free range)、MluCI、MluI §, MluI-HF(free strain), MlyI, MmeI, MnlI, MscI, MseI, MslI, MspA1I, MspI, MspJI, M woI, NaeI, NarI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, NciI, NcoI §, NcoI-HF(free strain), NdeI, NgoMIV, NheI-HF (free strain), NlaIII, NlaIV, NmeAIII, NotI §, NotI-HF (free strain), NruI §、NruI-HF(flower chain)、NsiI §、NsiI-HF(free-flower), NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaeR7I, PaqCI, P ciI, PflFI, PflMI, PI-PspI, PI-SceI, PleI, PluTI, PmeI, PmlI, PpuMI, PshAI, PsiI-v2, PspGI, PspOMI, PspXI, and PstI §、PstI-HF(free chain) PvuI §、PvuI-HF(chain chain) PvuII §、PvuII-HF(chain chain) RsaI RsrII SacI §、SacI-HF(free strain) SacII SalI §、SalI-HF(unfree strain) SapI Sau3AI Sau96I SbfI §、SbfI-HF(free radical), ScaI-HF(free radical), ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI §、SpeI-HF(high fuel)、SphI§, SphI-HF®, SrfI, SspI §, SspI-HF®, StuI, StyD4I, StyI-HF®, SwaI, TaqI-v2, TfiI, TseI, Tsp45I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI, or ZraI restriction cleavage sites.

[0022] In some embodiments, the nucleic acid stuffer does not encode an open reading frame (ORF) larger than 10, 20, 30, 40, or 50 amino acids. In some embodiments, the nucleic acid stuffer does not encode an ORF larger than 20 amino acids.

[0023] In some embodiments, the nucleic acid stuffer sequence has a length of about 100 to about 5000 nucleic acid bases. In some embodiments, the nucleic acid stuffer sequence has a length of about 100 to about 5000, 100 to about 4000, 100 to about 3000, 100 to about 2000, 100 to about 1000, 100 to about 900, 100 to about 800, 100 to about 700, 100 to about 600, 100 to about 500, or 100 to about 400 nucleic acid bases. In some embodiments, the nucleic acid stuffer sequence has a length of about 100 to about 5000, 500 to about 5000, 1000 to about 5000, 2000 to about 5000, or 3000 to about 5000 nucleic acid bases. In some embodiments, the nucleic acid stuffer sequence has a length of about 1000 to about 5000, 1050 to about 4500, or 2000 to about 3000 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 3028 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 2235 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 381 nucleobases.

[0024] In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8. In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 100 nucleotides of SEQ ID NO: 7, 8, or 11.

[0025] In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence complementary to SEQ ID NO: 7. In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence complementary to SEQ ID NO:8. In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence complementary to SEQ ID NO: 11. In some embodiments, the nucleic acid stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence complementary to at least 100 nucleotides of SEQ ID NO: 7, 8, or 11.

[0026] In some embodiments, the nucleic acid stuffer sequence is selected from the group consisting of bases 1-100, 2-101, 3-102, 4-103, 5-104, 6-105, 7-106, 8-107, 9-108, 10-109, 11-110, 12-111, 13-112, 14-113, 15-114, 16-115, 17-116, 18-117, 19-118, 20-119, 21-120, 22-121, 23-122, 24-123, 25-124, 26-125, 27-126, 28-127, 29-128, 30-129, 31-130, 32-131, 33-132, 34-133, 35-134, 36-135, 37-136, 38-137, 39-138, 40-139, 41-140, 42-141, 43-142, 44-143, 45-144, 46-145, 47-146, 48-14 7, 49-148, 50-149, 51-150, 52-151, 53-152, 54-153, 55-154, 56-155, 57-156, 58-157, 59-158, 60-159, 61-160, 62-161, 63-162, 64-163, 65-164, 66-1 65, 67~166, 68~167, 69~168, 70~169, 71~170, 72~171, 73~172, 74~173, 75~174, 76~175, 77~176, 78~177, 79~178, 80~179, 81~180, 82~181, 83~182, 84 ~183, 85~184, 86~185, 87~186, 88~187, 89~188, 90~189, 91~190, 92~191, 93~192, 94~193, 95~194, 96~195, 97~196, 98~197, 99~198, 100~199, 101~200 , 102-201, 103-202, 104-203, 105-204, 106-205, 107-206, 108-207, 109-208, 110-209, 111-210, 112-211, 113-212, 114-213, 115-214, 116-215, 117 ~216, 118~217, 119~218, 120~219, 121~220, 122~221, 123~222, 124~223, 125~224, 126~225, 127~226, 128~227, 129~228, 130~229, 131~230, 132~231,133~232、134~233、135~234、136~235、137~236、138~237、139~238、140~239、141~240、142~241、143~242、144~243、145~244、146~245、147~246、148~247、149~248、150~249、151~250、152~251、153~252、154~253、155~254、156~255、157~256、158~257、159~258、160~259、161~260、162~261、163~262、164~263、165~264、166~265、167~266、168~267、169~268、170~269、171~270、172~271、173~272、174~273、175~274、176~275、177~276、178~277、179~278、180~279、181~280、182~281、183~282、184~283、185~284、186~285、187~286、188~287、189~288、190~289、191~290、192~291、193~292、194~293、195~294、196~295、197~296、198~297、199~298、200~299、201~300、202~301、203~302、204~303、205~304、206~305、207~306、208~307、209~308、210~309、211~310、212~311、213~312、214~313、215~314、216~315、217~316、218~317、219~318、220~319、221~320、222~321、223~322、224~323、225~324、226~325、227~326、228~327、229~328、230~329、231~330、232~331、233~332、234~333、235~334、236~335、237~336、238~337、239~338、240~339、241~340、242~341、243~342、244~343、245~344、246~345、247~346、248~347、249~348、250~349、251~350、252~351、253~352、254~353、255~354、256~355、257~356、258~357、259~358、260~359、261~360、262~361、263~362、264~363、265~364、266~365、267~366、268~367、269~368、270~369、271~370、272~371、273~372、274~373、275~374、276~375、277~376、278~377、279~378、280~379、281~380、282~381、283~382、284~383、285~384、286~385、287~386、288~387、289~388、290~389、291~390、292~391、293~392、294~393、295~394、296~395、297~396、298~397、299~398、300~399、301~400、302~401、303~402、304~403、305~404、306~405、307~406、308~407、309~408、310~409、311~410、312~411、313~412、314~413、315~414、316~415、317~416、318~417、319~418、320~419、321~420、322~421、323~422、324~423、325~424、326~425、327~426、328~427、329~428、330~429、331~430、332~431、333~432、334~433、335~434、336~435、337~436、338~437、339~438、340~439、341~440、342~441、343~442、344~443、345~444、346~445、347~446、348~447、349~448、350~449、351~450、352~451、353~452、354~453、355~454、356~455、357~456、358~457、359~458、360~459、361~460、362~461、363~462、364~463、365~464、366~465、367~466、368~467、369~468、370~469、371~470、372~471、373~472、374~473、375~474、376~475、377~476、378~477、379~478、380~479、381~480、382~481、383~482、384~483、385~484、386~485、387~486、388~487、389~488、390~489、391~490、392~491、393~492、394~493、395~494、396~495、397~496、398~497、399~498、400~499、401~500、402~501、403~502、404~503、405~504、406~505、407~506、408~507、409~508、410~509、411~510、412~511、413~512、414~513、415~514、416~515、417~516、418~517、419~518、420~519、421~520、422~521、423~522、424~523、425~524、426~525、427~526、428~527、429~528、430~529、431~530、432~531、433~532、434~533、435~534、436~535、437~536、438~537、439~538、440~539、441~540、442~541、443~542、444~543、445~544、446~545、447~546、448~547、449~548、450~549、451~550、452~551、453~552、454~553、455~554、456~555、457~556、458~557、459~558、460~559、461~560、462~561、463~562、464~563、465~564、466~565、467~566、468~567、469~568、470~569、471~570、472~571、473~572、474~573、475~574、476~575、477~576、478~577、479~578、480~579、481~580、482~581、483~582、484~583、485~584、486~585、487~586、488~587、489~588、490~589、491~590、492~591、493~592、494~593、495~594、496~595、497~596、498~597、499~598、500~599、501~600、502~601、503~602、504~603、505~604、506~605、507~606、508~607、509~608、510~609、511~610、512~611、513~612、514~613、515~614、516~615、517~616、518~617、519~618、520~619、521~620、522~621、523~622、524~623、525~624、526~625、527~626、528~627、529~628、530~629、531~630、532~631、533~632、534~633、535~634、536~635、537~636、538~637、539~638、540~639、541~640、542~641、543~642、544~643、545~644、546~645、547~646、548~647、549~648、550~649、551~650、552~651、553~652、554~653、555~654、556~655、557~656、558~657、559~658、560~659、561~660、562~661、563~662、564~663、565~664、566~665、567~666、568~667、569~668、570~669、571~670、572~671、573~672、574~673、575~674、576~675、577~676、578~677、579~678、580~679、581~680、582~681、583~682、584~683、585~684、586~685、587~686、588~687、589~688、590~689、591~690、592~691、593~692、594~693、595~694、596~695、597~696、598~697、599~698、600~699、601~700、602~701、603~702、604~703、605~704、606~705、607~706、608~707、609~708、610~709、611~710、612~711、613~712、614~713、615~714、616~715、617~716、618~717、619~718、620~719、621~720、622~721、623~722、624~723、625~724、626~725、627~726、628~727、629~728、630~729、631~730、632~731、633~732、634~733、635~734、636~735、637~736、638~737、639~738、640~739、641~740、642~741、643~742、644~743、645~744、646~745、647~746、648~、 747、649~748、650~749、651~750、652~751、653~752、654~753、655~754、656~755、657~756、658~757、659~758、660~759、661~760、662~761、663~762、664~763、665~764、666~765、667~766、668~767、669~768、670~769、671~770、672~771、673~772、674~773、675~774、676~775、677~776、678~777、679~778、680~779、681~780、682~781、683~782、684~783、685~784、686~785、687~786、688~787、689~788、690~789、691~790、692~791、693~792、694~793、695~794、696~795、697~796、698~797、699~798、700~799、701~800、702~801、703~802、704~803、705~804、706~805、707~806、708~807、709~808、710~809、711~810、712~811、713~812、714~813、715~814、716~815、717~816、718~817、719~818、720~819、721~820、722~821、723~822、724~823、725~824、726~825、727~826、728~827、729~828、730~829、731~830、732~831、733~832、734~833、735~834、736~835、737~836、738~837、739~838、740~839、741~840、742~841、743~842、744~843、745~844、746~845、747~846、748~847、749~848、750~849、751~850、752~851、753~852、754~853、755~854、756~855、757~856、758~857、759~858、760~859、761~860、762~861、763~862、764~863、765~864、766~865、767~866、768~867、769~868、770~869、771~870、772~871、773~872、774~873、775~874、776~875、777~876、778~877、779~878、780~879、781~880、782~881、783~882、784~883、785~884、786~885、787~886、788~887、789~888、790~889、791~890、792~891、793~892、794~893、795~894、796~895、797~896、798~897、799~898、800~899、801~900、802~901、803~902、804~903、805~904、806~905、807~906、808~907、809~908、810~909、811~910、812~911、813~912、814~913、815~914、816~915、817~916、818~917、819~918、820~919、821~920、822~921、823~922、824~923、825~924、826~925、827~926、828~927、829~928、830~929、831~930、832~931、833~932、834~933、835~934、836~935、837~936、838~937、839~938、840~939、841~940、842~941、843~942、844~943、845~944、846~945、847~946、848~947、849~948、850~949、851~950、852~951、853~952、854~953、855~954、856~955、857~956、858~957、859~958、860~959、861~960、862~961、863~962、864~963、865~964、866~965、867~966、868~967、869~968、870~969、871~970、872~971、873~972、874~973、875~974、876~975、877~976、878~977、879~978、880~979、881~980、882~981、883~982、884~983、885~984、886~985、887~986、888~987、889~988、890~989、891~990、892~991、893~992、894~993、895~994、896~995、897~996、898~997、899~998、900~999、901~1000、902~1001、903~1002、904~1003、905~1004、906~1005、907~1006、908~1007、909~1008、910~1009、911~1010、912~1011、913~1012、914~1013、915~1014、916~1015、917~1016、918~1017、919~1018、920~1019、921~1020、922~1021、923~1022、924~1023、925~1024、926~1025、927~1026、928~1027、929~1028、930~1029、931~1030、932~1031、933~1032、934~1033、935~1034、936~1035、937~1036、938~1037、939~1038、940~1039、941~1040、942~1041、943~1042、944~1043、945~1044、946~1045、947~1046、948~1047、949~1048、950~1049、951~1050、952~1051、953~1052、954~1053、955~1054、956~1055、957~1056、958~1057、959~1058、960~1059、961~1060、962~1061、963~1062、964~1063、965~1064、966~1065、967~1066、968~1067、969~1068、970~1069、971~1070、972~1071、973~1072、974~1073、975~1074、976~1075、977~1076、978~1077、979~1078、980~1079、981~1080、982~1081、983~1082、984~1083、985~1084、986~1085、987~1086、988~1087、989~1088、990~1089、991~1090、992~1091、993~1092、994~1093、995~1094、996~1095、997~1096、998~1097、999~1098、1000~1099、1000~1100、1001~1101、1002~1102、1003~1103、1004~1104、1005~1105、1006~1106、1007~1107、1008~1108、1009~1109、1010~1110、1011~1111、1012~1112、1013~1113、1014~1114、1015~1115、1016~1116、1017~1117、1018~1118、1019~1119、1020~1120、1021~1121、1022~1122、1023~1123、1024~1124、1025~1125、1026~1126、1027~1127、1028~1128、1029~1129、1030~1130、1031~1131、1032~1132、1033~1133、1034~1134、1035~1135、1036~1136、1037~1137、1038~1138、1039~1139、1040~1140、1041~1141、1042~1142、1043~1143、1044~1144、1045~1145、1046~1146、1047~1147、1048~1148、1049~1149、1050~1150、1051~1151、1052~1152、1053~1153、1054~1154、1055~1155、1056~1156、1057~1157、1058~1158、1059~1159、1060~1160、1061~1161、1062~1162、1063~1163、1064~1164、1065~1165、1066~1166、1067~1167、1068~1168、1069~1169、1070~1170、1071~1171、1072~1172、1073~1173、1074~1174、1075~1175、1076~1176、1077~1177、1078~1178、1079~1179、1080~1180、1081~1181、1082~1182、1083~1183、1084~1184、1085~1185、1086~1186、1087~1187、1088~1188、1089~1189、1090~1190、1091~1191、1092~1192、1093~1193、1094~1194、1095~1195、1096~1196、1097~1197、1098~1198、1099~1199、1100~1200、1101~1201、1102~1202、1103~1203、1104~1204、1105~1205、1106~1206、1107~1207、1108~1208、1109~1209、1110~1210、1111~1211、1112~1212、1113~1213、1114~1214、1115~1215、1116~1216、1117~1217、1118~1218、1119~1219、1120~1220、1121~1221、1122~1222、1123~1223、1124~1224、1125~1225、1126~1226、1127~1227、1128~1228、1129~1229、1130~1230、1131~1231、1132~1232、1133~1233、1134~1234、1135~1235、1136~1236、1137~1237、1138~1238、1139~1239、1140~1240、1141~1241、1142~1242、1143~1243、1144~1244、1145~1245、1146~1246、1147~1247、1148~1248、1149~1249、1150~1250、1151~1251、1152~1252、1153~1253、1154~1254、1155~1255、1156~1256、1157~1257、1158~1258、1159~1259、1160~1260、1161~1261、1162~1262、1163~1263、1164~1264、1165~1265、1166~1266、1167~1267、1168~1268、1169~1269、1170~1270、1171~1271、1172~1272、1173~1273、1174~1274、1175~1275、1176~1276、1177~1277、1178~1278、1179~1279、1180~1280、1181~1281、1182~1282、1183~1283、1184~1284、1185~1285、1186~1286、1187~1287、1188~1288、1189~1289、1190~1290、1191~1291、1192~1292、1193~1293、1194~1294、1195~1295、1196~1296、1197~1297、1198~1298、1199~1299、1200~1300、1201~1301、1202~1302、1203~1303、1204~1304、1205~1305、1206~1306、1207~1307、1208~1308、1209~1309、1210~1310、1211~1311、1212~1312、1213~1313、1214~1314、1215~1315、1216~1316、1217~1317、1218~1318、1219~1319、 1220~1320、1221~1321、1222~1322、1223~1323、1224~1324、1225~1325、1226~1326、1227~1327、1228~1328、1229~1329、1230~1330、1231~1331、1232~1332、1233~1333、1234~1334、1235~1335、1236~1336、1237~1337、1238~1338、1239~1339、1240~1340、1241~1341、1242~1342、1243~1343、1244~1344、1245~1345、1246~1346、1247~1347、1248~1348、1249~1349、1250~1350、1251~1351、1252~1352、1253~1353、1254~1354、1255~1355、1256~1356、1257~1357、1258~1358、1259~1359、1260~1360、1261~1361、1262~1362、1263~1363、1264~1364、1265~1365、1266~1366、1267~1367、1268~1368、1269~1369、1270~1370、1271~1371、1272~1372、1273~1373、1274~1374、1275~1375、1276~1376、1277~1377、1278~1378、1279~1379、1280~1380、1281~1381、1282~1382、1283~1383、1284~1384、1285~1385、1286~1386、1287~1387、1288~1388、1289~1389、1290~1390、1291~1391、1292~1392、1293~1393、1294~1394、1295~1395、1296~1396、1297~1397、1298~1398、1299~1399、1300~1400、1301~1401、1302~1402、1303~1403、1304~1404、1305~1405、1306~1406、1307~1407、1308~1408、1309~1409、1310~1410、1311~1411、1312~1412、1313~1413、1314~1414、1315~1415、1316~1416、1317~1417、1318~1418、1319~1419、1320~1420、1321~1421、1322~1422、1323~1423、1324~1424、1325~1425、1326~1426、1327~1427、1328~1428、1329~1429、1330~1430、1331~1431、1332~1432、1333~1433、1334~1434、1335~1435、1336~1436、1337~1437、1338~1438、1339~1439、1340~1440、1341~1441、1342~1442、1343~1443、1344~1444、1345~1445、1346~1446、1347~1447、1348~1448、1349~1449、1350~1450、1351~1451、1352~1452、1353~1453、1354~1454、1355~1455、1356~1456、1357~1457、1358~1458、1359~1459、1360~1460、1361~1461、1362~1462、1363~1463、1364~1464、1365~1465、1366~1466、1367~1467、1368~1468、1369~1469、1370~1470、1371~1471、1372~1472、1373~1473、1374~1474、1375~1475、1376~1476、1377~1477、1378~1478、1379~1479、1380~1480、1381~1481、1382~1482、1383~1483、1384~1484、1385~1485、1386~1486、1387~1487、1388~1488、1389~1489、1390~1490、1391~1491、1392~1492、1393~1493、1394~1494、1395~1495、1396~1496、1397~1497、1398~1498、1399~1499、1400~1500、1401~1501、1402~1502、1403~1503、1404~1504、1405~1505、1406~1506、1407~1507、1408~1508、1409~1509、1410~1510、1411~1511、1412~1512、1413~1513、1414~1514、1415~1515、1416~1516、1417~1517、1418~1518、1419~1519、1420~1520、1421~1521、1422~1522、1423~1523、1424~1524、1425~1525、1426~1526、1427~1527、1428~1528、1429~1529、1430~1530、1431~1531、1432~1532、1433~1533、1434~1534、1435~1535、1436~1536、1437~1537、1438~1538、1439~1539、1440~1540、1441~1541、1442~1542、1443~1543、1444~1544、1445~1545、1446~1546、1447~1547、1448~1548、1449~1549、1450~1550、1451~1551、1452~1552、1453~1553、1454~1554、1455~1555、1456~1556、1457~1557、1458~1558、1459~1559、1460~1560、1461~1561、1462~1562、1463~1563、1464~1564、1465~1565、1466~1566、1467~1567、1468~1568、1469~1569、1470~1570、1471~1571、1472~1572、1473~1573、1474~1574、1475~1575、1476~1576、1477~1577、1478~1578、1479~1579、1480~1580、1481~1581、1482~1582、1483~1583、1484~1584、1485~1585、1486~1586、1487~1587、1488~1588、1489~1589、1490~1590、1491~1591、1492~1592、1493~1593、1494~1594、1495~1595、1496~1596、1497~1597、1498~1598、1499~1599、1500~1600、1501~1601、1502~1602、1503~1603、1504~1604、1505~1605、1506~1606、1507~1607、1508~1608、1509~1609、1510~1610、1511~1611、1512~1612、1513~1613、1514~1614、1515~1615、1516~1616、1517~1617、1518~1618、1519~1619、1520~1620、1521~1621、1522~1622、1523~1623、1524~1624、1525~1625、1526~1626、1527~1627、1528~1628、1529~1629、1530~1630、1531~1631、1532~1632、1533~1633、1534~1634、1535~1635、1536~1636、1537~1637、1538~1638、1539~1639、1540~1640、1541~1641、1542~1642、1543~1643、1544~1644、1545~1645、1546~1646、1547~1647、1548~1648、1549~1649、1550~1650、1551~1651、1552~1652、1553~1653、1554~1654、1555~1655、1556~1656、1557~1657、1558~1658、1559~1659、1560~1660、1561~1661、1562~1662、1563~1663、1564~1664、1565~1665、1566~1666、1567~1667、1568~1668、1569~1669、1570~1670、1571~1671、1572~1672、1573~1673、1574~1674、1575~1675、1576~1676、1577~1677、1578~1678、1579~1679、1580~1680、1581~1681、1582~1682、1583~1683、1584~1684、1585~1685、1586~1686、1587~1687、1588~1688、1589~1689、1590~1690、1591~1691、1592~1692、1593~1693、1594~1694、1595~1695、1596~1696、1597~1697、1598~1698、1599~1699、1600~1700、1601~1701、1602~1702、1603~1703、1604~1704、1605~1705、1606~1706、1607~1707、1608~1708、1609~1709、1610~1710、1611~1711、1612~1712、1613~1713、1614~1714、1615~1715、1616~1716、1617~1717、1618~1718、1619~1719、1620~1720、1621~1721、1622~1722、1623~1723、1624~1724、1625~1725、1626~1726、1627~1727、1628~1728、1629~1729、1630~1730、1631~1731、1632~1732、1633~1733、1634~1734、1635~1735、1636~1736、1637~1737、1638~1738、1639~1739、1640~1740、1641~1741、1642~1742、1643~1743、1644~1744、1645~1745、1646~1746、1647~1747、1648~1748、1649~1749、1650~1750、1651~1751、1652~1752、1653~1753、1654~1754、1655~1755、1656~1756、1657~1757、1658~1758、1659~1759、1660~1760、1661~1761、1662~1762、1663~1763、1664~1764、1665~1765、1666~1766、1667~1767、1668~1768、1669~1769、1670~1770、1671~1771、1672~1772、1673~1773、1674~1774、1675~1775、1676~1776、1677~1777、1678~1778、1679~1779、1680~1780、1681~1781、1682~1782、1683~1783、1684~1784、1685~1785、1686~1786、1687~1787、1688~1788、1689~1789、1690~1790、1691~1791、1692~1792、1693~1793、1694~1794、1695~1795、1696~1796、1697~1797、1698~1798、1699~1799、1700~1800、1701~1801、1702~1802、1703~1803、1704~1804、1705~1805、1706~1806、1707~1807、1708~1808、1709~1809、1710~1810、1711~1811、1712~1812、1713~1813、1714~1814、1715~1815、1716~1816、1717~1817、1718~1818、1719~1819、1720~1820、1721~1821、1722~1822、1723~1823、1724~1824、1725~1825、1726~1826、1727~1827、1728~1828、1729~1829、1730~1830、1731~1831、1、 732~1832、1733~1833、1734~1834、1735~1835、1736~1836、1737~1837、1738~1838、1739~1839、1740~1840、1741~1841、1742~1842、1743~1843、1744~1844、1745~1845、1746~1846、1747~1847、1748~1848、1749~1849、1750~1850、1751~1851、1752~1852、1753~1853、1754~1854、1755~1855、1756~1856、1757~1857、1758~1858、1759~1859、1760~1860、1761~1861、1762~1862、1763~1863、1764~1864、1765~1865、1766~1866、1767~1867、1768~1868、1769~1869、1770~1870、1771~1871、1772~1872、1773~1873、1774~1874、1775~1875、1776~1876、1777~1877、1778~1878、1779~1879、1780~1880、1781~1881、1782~1882、1783~1883、1784~1884、1785~1885、1786~1886、1787~1887、1788~1888、1789~1889、1790~1890、1791~1891、1792~1892、1793~1893、1794~1894、1795~1895、1796~1896、1797~1897、1798~1898、1799~1899、1800~1900、1801~1901、1802~1902、1803~1903、1804~1904、1805~1905、1806~1906、1807~1907、1808~1908、1809~1909、1810~1910、1811~1911、1812~1912、1813~1913、1814~1914、1815~1915、1816~1916、1817~1917、1818~1918、1819~1919、1820~1920、1821~1921、1822~1922、1823~1923、1824~1924、1825~1925、1826~1926、1827~1927、1828~1928、1829~1929、1830~1930、1831~1931、1832~1932、1833~1933、1834~1934、1835~1935、1836~1936、1837~1937、1838~1938、1839~1939、1840~1940、1841~1941、1842~1942、1843~1943、1844~1944、1845~1945、1846~1946、1847~1947、1848~1948、1849~1949、1850~1950、1851~1951、1852~1952、1853~1953、1854~1954、1855~1955、1856~1956、1857~1957、1858~1958、1859~1959、1860~1960、1861~1961、1862~1962、1863~1963、1864~1964、1865~1965、1866~1966、1867~1967、1868~1968、1869~1969、1870~1970、1871~1971、1872~1972、1873~1973、1874~1974、1875~1975、1876~1976、1877~1977、1878~1978、1879~1979、1880~1980、1881~1981、1882~1982、1883~1983、1884~1984、1885~1985、1886~1986、1887~1987、1888~1988、1889~1989、1890~1990、1891~1991、1892~1992、1893~1993、1894~1994、1895~1995、1896~1996、1897~1997、1898~1998、1899~1999、1900~2000、1901~2001、1902~2002、1903~2003、1904~2004、1905~2005、1906~2006、1907~2007、1908~2008、1909~2009、1910~2010、1911~2011、1912~2012、1913~2013、1914~2014、1915~2015、1916~2016、1917~2017、1918~2018、1919~2019、1920~2020、1921~2021、1922~2022、1923~2023、1924~2024、1925~2025、1926~2026、1927~2027、1928~2028、1929~2029、1930~2030、1931~2031、1932~2032、1933~2033、1934~2034、1935~2035、1936~2036、1937~2037、1938~2038、1939~2039、1940~2040、1941~2041、1942~2042、1943~2043、1944~2044、1945~2045、1946~2046、1947~2047、1948~2048、1949~2049、1950~2050、1951~2051、1952~2052、1953~2053、1954~2054、1955~2055、1956~2056、1957~2057、1958~2058、1959~2059、1960~2060、1961~2061、1962~2062、1963~2063、1964~2064、1965~2065、1966~2066、1967~2067、1968~2068、1969~2069、1970~2070、1971~2071、1972~2072、1973~2073、1974~2074、1975~2075、1976~2076、1977~2077、1978~2078、1979~2079、1980~2080、1981~2081、1982~2082、1983~2083、1984~2084、1985~2085、1986~2086、1987~2087、1988~2088、1989~2089、1990~2090、1991~2091、1992~2092、1993~2093、1994~2094、1995~2095、1996~2096、1997~2097、1998~2098、1999~2099、2000~2100、2001~2101、2002~2102、2003~2103、2004~2104、2005~2105、2006~2106、2007~2107、2008~2108、2009~2109、2010~2110、2011~2111、2012~2112、2013~2113、2014~2114、2015~2115、2016~2116、2017~2117、2018~2118、2019~2119、2020~2120、2021~2121、2022~2122、2023~2123、2024~2124、2025~2125、2026~2126、2027~2127、2028~2128、2029~2129、2030~2130、2031~2131、2032~2132、2033~2133、2034~2134、2035~2135、2036~2136、2037~2137、2038~2138、2039~2139、2040~2140、2041~2141、2042~2142、2043~2143、2044~2144、2045~2145、2046~2146、2047~2147、2048~2148、2049~2149、2050~2150、2051~2151、2052~2152、2053~2153、2054~2154、2055~2155、2056~2156、2057~2157、2058~2158、2059~2159、2060~2160、2061~2161、2062~2162、2063~2163、2064~2164、2065~2165、2066~2166、2067~2167、2068~2168、2069~2169、2070~2170、2071~2171、2072~2172、2073~2173、2074~2174、2075~2175、2076~2176、2077~2177、2078~2178、2079~2179、2080~2180、2081~2181、2082~2182、2083~2183、2084~2184、2085~2185、2086~2186、2087~2187、2088~2188、2089~2189、2090~2190、2091~2191、2092~2192、2093~2193、2094~2194、2095~2195、2096~2196、2097~2197、2098~2198、2099~2199、2100~2200、2101~2201、2102~2202、2103~2203、2104~2204、2105~2205、2106~2206、2107~2207、2108~2208、2109~2209、2110~2210、2111~2211、2112~2212、2113~2213、2114~2214、2115~2215、2116~2216、2117~2217、2118~2218、2119~2219、2120~2220、2121~2221、2122~2222、2123~2223、2124~2224、2125~2225、2126~2226、2127~2227、2128~2228、2129~2229、2130~2230、2131~2231、2132-2232, 2133-2233, 2134-2234, or 2135-2235, which are about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 2132-2232, 2133-2233, 2134-2234, or 2135-2235.

[0027] In some embodiments, the nucleic acid stuffer sequence is selected from the group consisting of bases 1-100, 2-101, 3-102, 4-103, 5-104, 6-105, 7-106, 8-107, 9-108, 10-109, 11-110, 12-111, 13-112, 14-113, 15-114, 16-115, 17-116, 18-117, 19-118, 20-119, 21-120, 22-121, 23-122, 24-123, 25-124, 26-125, 27-126, 28-127, 29-128, 30-129, 31-130, 32-131, 33-132, 34-133, 35-134, 36-135, 37-136, 38-137, 39-138, 40-139, 41-140, 42-141, 43-142, 44-143, 45-144, 46-145, 47-146, 48-14 7, 49-148, 50-149, 51-150, 52-151, 53-152, 54-153, 55-154, 56-155, 57-156, 58-157, 59-158, 60-159, 61-160, 62-161, 63-162, 64-163, 65-164, 66-1 65, 67~166, 68~167, 69~168, 70~169, 71~170, 72~171, 73~172, 74~173, 75~174, 76~175, 77~176, 78~177, 79~178, 80~179, 81~180, 82~181, 83~182, 84 ~183, 85~184, 86~185, 87~186, 88~187, 89~188, 90~189, 91~190, 92~191, 93~192, 94~193, 95~194, 96~195, 97~196, 98~197, 99~198, 100~199, 101~200 , 102-201, 103-202, 104-203, 105-204, 106-205, 107-206, 108-207, 109-208, 110-209, 111-210, 112-211, 113-212, 114-213, 115-214, 116-215, 117 ~216, 118~217, 119~218, 120~219, 121~220, 122~221, 123~222, 124~223, 125~224, 126~225, 127~226, 128~227, 129~228, 130~229, 131~230, 132~231,133~232、134~233、135~234、136~235、137~236、138~237、139~238、140~239、141~240、142~241、143~242、144~243、145~244、146~245、147~246、148~247、149~248、150~249、151~250、152~251、153~252、154~253、155~254、156~255、157~256、158~257、159~258、160~259、161~260、162~261、163~262、164~263、165~264、166~265、167~266、168~267、169~268、170~269、171~270、172~271、173~272、174~273、175~274、176~275、177~276、178~277、179~278、180~279、181~280、182~281、183~282、184~283、185~284、186~285、187~286、188~287、189~288、190~289、191~290、192~291、193~292、194~293、195~294、196~295、197~296、198~297、199~298、200~299、201~300、202~301、203~302、204~303、205~304、206~305、207~306、208~307、209~308、210~309、211~310、212~311、213~312、214~313、215~314、216~315、217~316、218~317、219~318、220~319、221~320、222~321、223~322、224~323、225~324、226~325、227~326、228~327、229~328、230~329、231~330、232~331、233~332、234~333、235~334、236~335、237~336、238~337、239~338、240~339、241~340、242~341、243~342、244~343、245~344、246~345、247~346、248~347、249~348、250~349、251~350、252~351、253~352、254~353、255~354、256~355、257~356、258~357、259~358、260~359、261~360、262~361、263~362、264~363、265~364、266~365、267~366、268~367、269~368、270~369、271~370、272~371、273~372、274~373、275~374、276~375、277~376、278~377、279~378、280~379、281~380、282~381、283~382、284~383、285~384、286~385、287~386、288~387、289~388、290~389、291~390、292~391、293~392、294~393、295~394、296~395、297~396、298~397、299~398、300~399、301~400、302~401、303~402、304~403、305~404、306~405、307~406、308~407、309~408、310~409、311~410、312~411、313~412、314~413、315~414、316~415、317~416、318~417、319~418、320~419、321~420、322~421、323~422、324~423、325~424、326~425、327~426、328~427、329~428、330~429、331~430、332~431、333~432、334~433、335~434、336~435、337~436、338~437、339~438、340~439、341~440、342~441、343~442、344~443、345~444、346~445、347~446、348~447、349~448、350~449、351~450、352~451、353~452、354~453、355~454、356~455、357~456、358~457、359~458、360~459、361~460、362~461、363~462、364~463、365~464、366~465、367~466、368~467、369~468、370~469、371~470、372~471、373~472、374~473、375~474、376~475、377~476、378~477、379~478、380~479、381~480、382~481、383~482、384~483、385~484、386~485、387~486、388~487、389~488、390~489、391~490、392~491、393~492、394~493、395~494、396~495、397~496、398~497、399~498、400~499、401~500、402~501、403~502、404~503、405~504、406~505、407~506、408~507、409~508、410~509、411~510、412~511、413~512、414~513、415~514、416~515、417~516、418~517、419~518、420~519、421~520、422~521、423~522、424~523、425~524、426~525、427~526、428~527、429~528、430~529、431~530、432~531、433~532、434~533、435~534、436~535、437~536、438~537、439~538、440~539、441~540、442~541、443~542、444~543、445~544、446~545、447~546、448~547、449~548、450~549、451~550、452~551、453~552、454~553、455~554、456~555、457~556、458~557、459~558、460~559、461~560、462~561、463~562、464~563、465~564、466~565、467~566、468~567、469~568、470~569、471~570、472~571、473~572、474~573、475~574、476~575、477~576、478~577、479~578、480~579、481~580、482~581、483~582、484~583、485~584、486~585、487~586、488~587、489~588、490~589、491~590、492~591、493~592、494~593、495~594、496~595、497~596、498~597、499~598、500~599、501~600、502~601、503~602、504~603、505~604、506~605、507~606、508~607、509~608、510~609、511~610、512~611、513~612、514~613、515~614、516~615、517~616、518~617、519~618、520~619、521~620、522~621、523~622、524~623、525~624、526~625、527~626、528~627、529~628、530~629、531~630、532~631、533~632、534~633、535~634、536~635、537~636、538~637、539~638、540~639、541~640、542~641、543~642、544~643、545~644、546~645、547~646、548~647、549~648、550~649、551~650、552~651、553~652、554~653、555~654、556~655、557~656、558~657、559~658、560~659、561~660、562~661、563~662、564~663、565~664、566~665、567~666、568~667、569~668、570~669、571~670、572~671、573~672、574~673、575~674、576~675、577~676、578~677、579~678、580~679、581~680、582~681、583~682、584~683、585~684、586~685、587~686、588~687、589~688、590~689、591~690、592~691、593~692、594~693、595~694、596~695、597~696、598~697、599~698、600~699、601~700、602~701、603~702、604~703、605~704、606~705、607~706、608~707、609~708、610~709、611~710、612~711、613~712、614~713、615~714、616~715、617~716、618~717、619~718、620~719、621~720、622~721、623~722、624~723、625~724、626~725、627~726、628~727、629~728、630~729、631~730、632~731、633~732、634~733、635~734、636~735、637~736、638~737、639~738、640~739、641~740、642~741、643~742、644~743、645~744、646~745、647~746、648~、 747、649~748、650~749、651~750、652~751、653~752、654~753、655~754、656~755、657~756、658~757、659~758、660~759、661~760、662~761、663~762、664~763、665~764、666~765、667~766、668~767、669~768、670~769、671~770、672~771、673~772、674~773、675~774、676~775、677~776、678~777、679~778、680~779、681~780、682~781、683~782、684~783、685~784、686~785、687~786、688~787、689~788、690~789、691~790、692~791、693~792、694~793、695~794、696~795、697~796、698~797、699~798、700~799、701~800、702~801、703~802、704~803、705~804、706~805、707~806、708~807、709~808、710~809、711~810、712~811、713~812、714~813、715~814、716~815、717~816、718~817、719~818、720~819、721~820、722~821、723~822、724~823、725~824、726~825、727~826、728~827、729~828、730~829、731~830、732~831、733~832、734~833、735~834、736~835、737~836、738~837、739~838、740~839、741~840、742~841、743~842、744~843、745~844、746~845、747~846、748~847、749~848、750~849、751~850、752~851、753~852、754~853、755~854、756~855、757~856、758~857、759~858、760~859、761~860、762~861、763~862、764~863、765~864、766~865、767~866、768~867、769~868、770~869、771~870、772~871、773~872、774~873、775~874、776~875、777~876、778~877、779~878、780~879、781~880、782~881、783~882、784~883、785~884、786~885、787~886、788~887、789~888、790~889、791~890、792~891、793~892、794~893、795~894、796~895、797~896、798~897、799~898、800~899、801~900、802~901、803~902、804~903、805~904、806~905、807~906、808~907、809~908、810~909、811~910、812~911、813~912、814~913、815~914、816~915、817~916、818~917、819~918、820~919、821~920、822~921、823~922、824~923、825~924、826~925、827~926、828~927、829~928、830~929、831~930、832~931、833~932、834~933、835~934、836~935、837~936、838~937、839~938、840~939、841~940、842~941、843~942、844~943、845~944、846~945、847~946、848~947、849~948、850~949、851~950、852~951、853~952、854~953、855~954、856~955、857~956、858~957、859~958、860~959、861~960、862~961、863~962、864~963、865~964、866~965、867~966、868~967、869~968、870~969、871~970、872~971、873~972、874~973、875~974、876~975、877~976、878~977、879~978、880~979、881~980、882~981、883~982、884~983、885~984、886~985、887~986、888~987、889~988、890~989、891~990、892~991、893~992、894~993、895~994、896~995、897~996、898~997、899~998、900~999、901~1000、902~1001、903~1002、904~1003、905~1004、906~1005、907~1006、908~1007、909~1008、910~1009、911~1010、912~1011、913~1012、914~1013、915~1014、916~1015、917~1016、918~1017、919~1018、920~1019、921~1020、922~1021、923~1022、924~1023、925~1024、926~1025、927~1026、928~1027、929~1028、930~1029、931~1030、932~1031、933~1032、934~1033、935~1034、936~1035、937~1036、938~1037、939~1038、940~1039、941~1040、942~1041、943~1042、944~1043、945~1044、946~1045、947~1046、948~1047、949~1048、950~1049、951~1050、952~1051、953~1052、954~1053、955~1054、956~1055、957~1056、958~1057、959~1058、960~1059、961~1060、962~1061、963~1062、964~1063、965~1064、966~1065、967~1066、968~1067、969~1068、970~1069、971~1070、972~1071、973~1072、974~1073、975~1074、976~1075、977~1076、978~1077、979~1078、980~1079、981~1080、982~1081、983~1082、984~1083、985~1084、986~1085、987~1086、988~1087、989~1088、990~1089、991~1090、992~1091、993~1092、994~1093、995~1094、996~1095、997~1096、998~1097、999~1098、1000~1099、1000~1100、1001~1101、1002~1102、1003~1103、1004~1104、1005~1105、1006~1106、1007~1107、1008~1108、1009~1109、1010~1110、1011~1111、1012~1112、1013~1113、1014~1114、1015~1115、1016~1116、1017~1117、1018~1118、1019~1119、1020~1120、1021~1121、1022~1122、1023~1123、1024~1124、1025~1125、1026~1126、1027~1127、1028~1128、1029~1129、1030~1130、1031~1131、1032~1132、1033~1133、1034~1134、1035~1135、1036~1136、1037~1137、1038~1138、1039~1139、1040~1140、1041~1141、1042~1142、1043~114、1044~1144、1045~1145、1046~1146、1047~1147、1048~1148、1049~1149、1050~1150、1051~1151、1052~1152、1053~1153、1054~1154、1055~1155、1056~1156、1057~1157、1058~1158、1059~1159、1060~1160、1061~1161、1062~1162、1063~1163、1064~1164、1065~1165、1066~1166、1067~1167、1068~1168、1069~1169、1070~1170、1071~1171、1072~1172、1073~1173、1074~1174、1075~1175、1076~1176、1077~1177、1078~1178、1079~1179、1080~1180、1081~1181、1082~1182、1083~1183、1084~1184、1085~1185、1086~1186、1087~1187、1088~1188、1089~1189、1090~1190、1091~1191、1092~1192、1093~1193、1094~1194、1095~1195、1096~1196、1097~1197、1098~1198、1099~1199、1100~1200、1101~1201、1102~1202、1103~1203、1104~1204、1105~1205、1106~1206、1107~1207、1108~1208、1109~1209、1110~1210、1111~1211、1112~1212、1113~1213、1114~1214、1115~1215、1116~1216、1117~1217、1118~1218、1119~1219、1120~1220、1121~1221、1122~1222、1123~1223、1124~1224、1125~1225、1126~1226、1127~1227、1128~1228、1129~1229、1130~1230、1131~1231、1132~1232、1133~1233、1134~1234、1135~1235、1136~1236、1137~1237、1138~1238、1139~1239、1140~1240、1141~1241、1142~1242、1143~1243、1144~1244、1145~1245、1146~1246、1147~1247、1148~1248、1149~1249、1150~1250、1151~1251、1152~1252、1153~1253、1154~1254、1155~1255、1156~1256、1157~1257、1158~1258、1159~1259、1160~1260、1161~1261、1162~1262、1163~1263、1164~1264、1165~1265、1166~1266、1167~1267、1168~1268、1169~1269、1170~1270、1171~1271、1172~1272、1173~1273、1174~1274、1175~1275、1176~1276、1177~1277、1178~1278、1179~1279、1180~1280、1181~1281、1182~1282、1183~1283、1184~1284、1185~1285、1186~1286、1187~1287、1188~1288、1189~1289、1190~1290、1191~1291、1192~1292、1193~1293、1194~1294、1195~1295、1196~1296、1197~1297、1198~1298、1199~1299、1200~1300、1201~1301、1202~1302、1203~1303、1204~1304、1205~1305、1206~1306、1207~1307、1208~1308、1209~1309、1210~1310、1211~1311、1212~1312、1213~1313、1214~1314、1215~1315、1216~1316、1217~1317、1218~1318、1219~1319、1、 220~1320、1221~1321、1222~1322、1223~1323、1224~1324、1225~1325、1226~1326、1227~1327、1228~1328、1229~1329、1230~1330、1231~1331、1232~1332、1233~1333、1234~1334、1235~1335、1236~1336、1237~1337、1238~1338、1239~1339、1240~1340、1241~1341、1242~1342、1243~1343、1244~1344、1245~1345、1246~1346、1247~1347、1248~1348、1249~1349、1250~1350、1251~1351、1252~1352、1253~1353、1254~1354、1255~1355、1256~1356、1257~1357、1258~1358、1259~1359、1260~1360、1261~1361、1262~1362、1263~1363、1264~1364、1265~1365、1266~1366、1267~1367、1268~1368、1269~1369、1270~1370、1271~1371、1272~1372、1273~1373、1274~1374、1275~1375、1276~1376、1277~1377、1278~1378、1279~1379、1280~1380、1281~1381、1282~1382、1283~1383、1284~1384、1285~1385、1286~1386、1287~1387、1288~1388、1289~1389、1290~1390、1291~1391、1292~1392、1293~1393、1294~1394、1295~1395、1296~1396、1297~1397、1298~1398、1299~1399、1300~1400、1301~1401、1302~1402、1303~1403、1304~1404、1305~1405、1306~1406、1307~1407、1308~1408、1309~1409、1310~1410、1311~1411、1312~1412、1313~1413、1314~1414、1315~1415、1316~1416、1317~1417、1318~1418、1319~1419、1320~1420、1321~1421、1322~1422、1323~1423、1324~1424、1325~1425、1326~1426、1327~1427、1328~1428、1329~1429、1330~1430、1331~1431、1332~1432、1333~1433、1334~1434、1335~1435、1336~1436、1337~1437、1338~1438、1339~1439、1340~1440、1341~1441、1342~1442、1343~1443、1344~1444、1345~1445、1346~1446、1347~1447、1348~1448、1349~1449、1350~1450、1351~1451、1352~1452、1353~1453、1354~1454、1355~1455、1356~1456、1357~1457、1358~1458、1359~1459、1360~1460、1361~1461、1362~1462、1363~1463、1364~1464、1365~1465、1366~1466、1367~1467、1368~1468、1369~1469、1370~1470、1371~1471、1372~1472、1373~1473、1374~1474、1375~1475、1376~1476、1377~1477、1378~1478、1379~1479、1380~1480、1381~1481、1382~1482、1383~1483、1384~1484、1385~1485、1386~1486、1387~1487、1388~1488、1389~1489、1390~1490、1391~1491、1392~1492、1393~1493、1394~1494、1395~1495、1396~1496、1397~1497、1398~1498、1399~1499、1400~1500、1401~1501、1402~1502、1403~1503、1404~1504、1405~1505、1406~1506、1407~1507、1408~1508、1409~1509、1410~1510、1411~1511、1412~1512、1413~1513、1414~1514、1415~1515、1416~1516、1417~1517、1418~1518、1419~1519、1420~1520、1421~1521、1422~1522、1423~1523、1424~1524、1425~1525、1426~1526、1427~1527、1428~1528、1429~1529、1430~1530、1431~1531、1432~1532、1433~1533、1434~1534、1435~1535、1436~1536、1437~1537、1438~1538、1439~1539、1440~1540、1441~1541、1442~1542、1443~1543、1444~1544、1445~1545、1446~1546、1447~1547、1448~1548、1449~1549、1450~1550、1451~1551、1452~1552、1453~1553、1454~1554、1455~1555、1456~1556、1457~1557、1458~1558、1459~1559、1460~1560、1461~1561、1462~1562、1463~1563、1464~1564、1465~1565、1466~1566、1467~1567、1468~1568、1469~1569、1470~1570、1471~1571、1472~1572、1473~1573、1474~1574、1475~1575、1476~1576、1477~1577、1478~1578、1479~1579、1480~1580、1481~1581、1482~1582、1483~1583、1484~1584、1485~1585、1486~1586、1487~1587、1488~1588、1489~1589、1490~1590、1491~1591、1492~1592、1493~1593、1494~1594、1495~1595、1496~1596、1497~1597、1498~1598、1499~1599、1500~1600、1501~1601、1502~1602、1503~1603、1504~1604、1505~1605、1506~1606、1507~1607、1508~1608、1509~1609、1510~1610、1511~1611、1512~1612、1513~1613、1514~1614、1515~1615、1516~1616、1517~1617、1518~1618、1519~1619、1520~1620、1521~1621、1522~1622、1523~1623、1524~1624、1525~1625、1526~1626、1527~1627、1528~1628、1529~1629、1530~1630、1531~1631、1532~1632、1533~1633、1534~1634、1535~1635、1536~1636、1537~1637、1538~1638、1539~1639、1540~1640、1541~1641、1542~1642、1543~1643、1544~1644、1545~1645、1546~1646、1547~1647、1548~1648、1549~1649、1550~1650、1551~1651、1552~1652、1553~1653、1554~1654、1555~1655、1556~1656、1557~1657、1558~1658、1559~1659、1560~1660、1561~1661、1562~1662、1563~1663、1564~1664、1565~1665、1566~1666、1567~1667、1568~1668、1569~1669、1570~1670、1571~1671、1572~1672、1573~1673、1574~1674、1575~1675、1576~1676、1577~1677、1578~1678、1579~1679、1580~1680、1581~1681、1582~1682、1583~1683、1584~1684、1585~1685、1586~1686、1587~1687、1588~1688、1589~1689、1590~1690、1591~1691、1592~1692、1593~1693、1594~1694、1595~1695、1596~1696、1597~1697、1598~1698、1599~1699、1600~1700、1601~1701、1602~1702、1603~1703、1604~1704、1605~1705、1606~1706、1607~1707、1608~1708、1609~1709、1610~1710、1611~1711、1612~1712、1613~1713、1614~1714、1615~1715、1616~1716、1617~1717、1618~1718、1619~1719、1620~1720、1621~1721、1622~1722、1623~1723、1624~1724、1625~1725、1626~1726、1627~1727、1628~1728、1629~1729、1630~1730、1631~1731、1632~1732、1633~1733、1634~1734、1635~1735、1636~1736、1637~1737、1638~1738、1639~1739、1640~1740、1641~1741、1642~1742、1643~1743、1644~1744、1645~1745、1646~1746、1647~1747、1648~1748、1649~1749、1650~1750、1651~1751、1652~1752、1653~1753、1654~1754、1655~1755、1656~1756、1657~1757、1658~1758、1659~1759、1660~1760、1661~1761、1662~1762、1663~1763、1664~1764、1665~1765、1666~1766、1667~1767、1668~1768、1669~1769、1670~1770、1671~1771、1672~1772、1673~1773、1674~1774、1675~1775、1676~1776、1677~1777、1678~1778、1679~1779、1680~1780、1681~1781、1682~1782、1683~1783、1684~1784、1685~1785、1686~1786、1687~1787、1688~1788、1689~1789、1690~1790、1691~1791、1692~1792、1693~1793、1694~1794、1695~1795、1696~1796、1697~1797、1698~1798、1699~1799、1700~1800、1701~1801、1702~1802、1703~1803、1704~1804、1705~1805、1706~1806、1707~1807、1708~1808、1709~1809、1710~1810、1711~1811、1712~1812、1713~1813、1714~1814、1715~1815、1716~1816、1717~1817、1718~1818、1719~1819、1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32~1832、1733~1833、1734~1834、1735~1835、1736~1836、1737~1837、1738~1838、1739~1839、1740~1840、1741~1841、1742~1842、1743~1843、1744~1844、1745~1845、1746~1846、1747~1847、1748~1848、1749~1849、1750~1850、1751~1851、1752~1852、1753~1853、1754~1854、1755~1855、1756~1856、1757~1857、1758~1858、1759~1859、1760~1860、1761~1861、1762~1862、1763~1863、1764~1864、1765~1865、1766~1866、1767~1867、1768~1868、1769~1869、1770~1870、1771~1871、1772~1872、1773~1873、1774~1874、1775~1875、1776~1876、1777~1877、1778~1878、1779~1879、1780~1880、1781~1881、1782~1882、1783~1883、1784~1884、1785~1885、1786~1886、1787~1887、1788~1888、1789~1889、1790~1890、1791~1891、1792~1892、1793~1893、1794~1894、1795~1895、1796~1896、1797~1897、1798~1898、1799~1899、1800~1900、1801~1901、1802~1902、1803~1903、1804~1904、1805~1905、1806~1906、1807~1907、1808~1908、1809~1909、1810~1910、1811~1911、1812~1912、1813~1913、1814~1914、1815~1915、1816~1916、1817~1917、1818~1918、1819~1919、1820~1920、1821~1921、1822~1922、1823~1923、1824~1924、1825~1925、1826~1926、1827~1927、1828~1928、1829~1929、1830~1930、1831~1931、1832~1932、1833~1933、1834~1934、1835~1935、1836~1936、1837~1937、1838~1938、1839~1939、1840~1940、1841~1941、1842~1942、1843~1943、1844~1944、1845~1945、1846~1946、1847~1947、1848~1948、1849~1949、1850~1950、1851~1951、1852~1952、1853~1953、1854~1954、1855~1955、1856~1956、1857~1957、1858~1958、1859~1959、1860~1960、1861~1961、1862~1962、1863~1963、1864~1964、1865~1965、1866~1966、1867~1967、1868~1968、1869~1969、1870~1970、1871~1971、1872~1972、1873~1973、1874~1974、1875~1975、1876~1976、1877~1977、1878~1978、1879~1979、1880~1980、1881~1981、1882~1982、1883~1983、1884~1984、1885~1985、1886~1986、1887~1987、1888~1988、1889~1989、1890~1990、1891~1991、1892~1992、1893~1993、1894~1994、1895~1995、1896~1996、1897~1997、1898~1998、1899~1999、1900~2000、1901~2001、1902~2002、1903~2003、1904~2004、1905~2005、1906~2006、1907~2007、1908~2008、1909~2009、1910~2010、1911~2011、1912~2012、1913~2013、1914~2014、1915~2015、1916~2016、1917~2017、1918~2018、1919~2019、1920~2020、1921~2021、1922~2022、1923~2023、1924~2024、1925~2025、1926~2026、1927~2027、1928~2028、1929~2029、1930~2030、1931~2031、1932~2032、1933~2033、1934~2034、1935~2035、1936~2036、1937~2037、1938~2038、1939~2039、1940~2040、1941~2041、1942~2042、1943~2043、1944~2044、1945~2045、1946~2046、1947~2047、1948~2048、1949~2049、1950~2050、1951~2051、1952~2052、1953~2053、1954~2054、1955~2055、1956~2056、1957~2057、1958~2058、1959~2059、1960~2060、1961~2061、1962~2062、1963~2063、1964~2064、1965~2065、1966~2066、1967~2067、1968~2068、1969~2069、1970~2070、1971~2071、1972~2072、1973~2073、1974~2074、1975~2075、1976~2076、1977~2077、1978~2078、1979~2079、1980~2080、1981~2081、1982~2082、1983~2083、1984~2084、1985~2085、1986~2086、1987~2087、1988~2088、1989~2089、1990~2090、1991~2091、1992~2092、1993~2093、1994~2094、1995~2095、1996~2096、1997~2097、1998~2098、1999~2099、2000~2100、2001~2101、2002~2102、2003~2103、2004~2104、2005~2105、2006~2106、2007~2107、2008~2108、2009~2109、2010~2110、2011~2111、2012~2112、2013~2113、2014~2114、2015~2115、2016~2116、2017~2117、2018~2118、2019~2119、2020~2120、2021~2121、2022~2122、2023~2123、2024~2124、2025~2125、2026~2126、2027~2127、2028~2128、2029~2129、2030~2130、2031~2131、2032~2132、2033~2133、2034~2134、2035~2135、2036~2136、2037~2137、2038~2138、2039~2139、2040~2140、2041~2141、2042~2142、2043~2143、2044~2144、2045~2145、2046~2146、2047~2147、2048~2148、2049~2149、2050~2150、2051~2151、2052~2152、2053~2153、2054~2154、2055~2155、2056~2156、2057~2157、2058~2158、2059~2159、2060~2160、2061~2161、2062~2162、2063~2163、2064~2164、2065~2165、2066~2166、2067~2167、2068~2168、2069~2169、2070~2170、2071~2171、2072~2172、2073~2173、2074~2174、2075~2175、2076~2176、2077~2177、2078~2178、2079~2179、2080~2180、2081~2181、2082~2182、2083~2183、2084~2184、2085~2185、2086~2186、2087~2187、2088~2188、2089~2189、2090~2190、2091~2191、2092~2192、2093~2193、2094~2194、2095~2195、2096~2196、2097~2197、2098~2198、2099~2199、2100~2200、2101~2201、2102~2202、2103~2203、2104~2204、2105~2205、2106~2206、2107~2207、2108~2208、2109~2209、2110~2210、2111~2211、2112~2212、2113~2213、2114~2214、2115~2215、2116~2216、2117~2217、2118~2218、2119~2219、2120~2220、2121~2221、2122~2222、2123~2223、2124~2224、2125~2225、2126~2226、2127~2227、2128~2228、2129~2229、2130~2230、2131~2231、2132~2232、2133~2233、2134~2234、2135~2235、2136~2236、2137~2237、2138~2238、2139~2239、2140~2240、2141~2241、2142~2242、2143~2243、2144~2244、2145~2245、2146~2246、2147~2247、2148~2248、2149~2249、2150~2250、2151~2251、2152~2252、2153~2253、2154~2254、2155~2255、2156~2256、2157~2257、2158~2258、2159~2259、2160~2260、2161~2261、2162~2262、2163~2263、2164~2264、2165~2265、2166~2266、2167~2267、2168~2268、2169~2269、2170~2270、2171~2271、2172~2272、2173~2273、2174~2274、2175~2275、2176~2276、2177~2277、2178~2278、2179~2279、2180~2280、2181~2281、2182~2282、2183~2283、2184~2284、2185~2285、2186~2286、2187~2287、2188~2288、2189~2289、2190~2290、2191~2291、2192~2292、2193~2293、2194~2294、2195~2295、2196~2296、2197~2297、2198~2298、2199~2299、2200~2300、2201~2301、2202~2302、2203~2303、2204~2304、2205~2305、2206~2306、2207~2307、2208~2308、2209~2309、2210~2310、2211~2311、2212~2312、2213~2313、2214~2314、2215~2315、2216~2316、2217~2317、2218~2318、2219~2319、2220~2320、2221~2321、2222~2322、2223~2323、2224~2324、2225~2325、2226~2326、2227~2327、2228~2328、2229~2329、2230~2330、2231~2331、2232~2332、2233~2333、2234~2334、2235~2335、2236~2336、2237~2337、2238~2338、2239~2339、2240~2340、2241~2341、2242~2342、2243~2343、224、 4~2344、2245~2345、2246~2346、2247~2347、2248~2348、2249~2349、2250~2350、2251~2351、2252~2352、2253~2353、2254~2354、2255~2355、2256~2356、2257~2357、2258~2358、2259~2359、2260~2360、2261~2361、2262~2362、2263~2363、2264~2364、2265~2365、2266~2366、2267~2367、2268~2368、2269~2369、2270~2370、2271~2371、2272~2372、2273~2373、2274~2374、2275~2375、2276~2376、2277~2377、2278~2378、2279~2379、2280~2380、2281~2381、2282~2382、2283~2383、2284~2384、2285~2385、2286~2386、2287~2387、2288~2388、2289~2389、2290~2390、2291~2391、2292~2392、2293~2393、2294~2394、2295~2395、2296~2396、2297~2397、2298~2398、2299~2399、2300~2400、2301~2401、2302~2402、2303~2403、2304~2404、2305~2405、2306~2406、2307~2407、2308~2408、2309~2409、2310~2410、2311~2411、2312~2412、2313~2413、2314~2414、2315~2415、2316~2416、2317~2417、2318~2418、2319~2419、2320~2420、2321~2421、2322~2422、2323~2423、2324~2424、2325~2425、2326~2426、2327~2427、2328~2428、2329~2429、2330~2430、2331~2431、2332~2432、2333~2433、2334~2434、2335~2435、2336~2436、2337~2437、2338~2438、2339~2439、2340~2440、2341~2441、2342~2442、2343~2443、2344~2444、2345~2445、2346~2446、2347~2447、2348~2448、2349~2449、2350~2450、2351~2451、2352~2452、2353~2453、2354~2454、2355~2455、2356~2456、2357~2457、2358~2458、2359~2459、2360~2460、2361~2461、2362~2462、2363~2463、2364~2464、2365~2465、2366~2466、2367~2467、2368~2468、2369~2469、2370~2470、2371~2471、2372~2472、2373~2473、2374~2474、2375~2475、2376~2476、2377~2477、2378~2478、2379~2479、2380~2480、2381~2481、2382~2482、2383~2483、2384~2484、2385~2485、2386~2486、2387~2487、2388~2488、2389~2489、2390~2490、2391~2491、2392~2492、2393~2493、2394~2494、2395~2495、2396~2496、2397~2497、2398~2498、2399~2499、2400~2500、2401~2501、2402~2502、2403~2503、2404~2504、2405~2505、2406~2506、2407~2507、2408~2508、2409~2509、2410~2510、2411~2511、2412~2512、2413~2513、2414~2514、2415~2515、2416~2516、2417~2517、2418~2518、2419~2519、2420~2520、2421~2521、2422~2522、2423~2523、2424~2524、2425~2525、2426~2526、2427~2527、2428~2528、2429~2529、2430~2530、2431~2531、2432~2532、2433~2533、2434~2534、2435~2535、2436~2536、2437~2537、2438~2538、2439~2539、2440~2540、2441~2541、2442~2542、2443~2543、2444~2544、2445~2545、2446~2546、2447~2547、2448~2548、2449~2549、2450~2550、2451~2551、2452~2552、2453~2553、2454~2554、2455~2555、2456~2556、2457~2557、2458~2558、2459~2559、2460~2560、2461~2561、2462~2562、2463~2563、2464~2564、2465~2565、2466~2566、2467~2567、2468~2568、2469~2569、2470~2570、2471~2571、2472~2572、2473~2573、2474~2574、2475~2575、2476~2576、2477~2577、2478~2578、2479~2579、2480~2580、2481~2581、2482~2582、2483~2583、2484~2584、2485~2585、2486~2586、2487~2587、2488~2588、2489~2589、2490~2590、2491~2591、2492~2592、2493~2593、2494~2594、2495~2595、2496~2596、2497~2597、2498~2598、2499~2599、2500~2600、2501~2601、2502~2602、2503~2603、2504~2604、2505~2605、2506~2606、2507~2607、2508~2608、2509~2609、2510~2610、2511~2611、2512~2612、2513~2613、2514~2614、2515~2615、2516~2616、2517~2617、2518~2618、2519~2619、2520~2620、2521~2621、2522~2622、2523~2623、2524~2624、2525~2625、2526~2626、2527~2627、2528~2628、2529~2629、2530~2630、2531~2631、2532~2632、2533~2633、2534~2634、2535~2635、2536~2636、2537~2637、2538~2638、2539~2639、2540~2640、2541~2641、2542~2642、2543~2643、2544~2644、2545~2645、2546~2646、2547~2647、2548~2648、2549~2649、2550~2650、2551~2651、2552~2652、2553~2653、2554~2654、2555~2655、2556~2656、2557~2657、2558~2658、2559~2659、2560~2660、2561~2661、2562~2662、2563~2663、2564~2664、2565~2665、2566~2666、2567~2667、2568~2668、2569~2669、2570~2670、2571~2671、2572~2672、2573~2673、2574~2674、2575~2675、2576~2676、2577~2677、2578~2678、2579~2679、2580~2680、2581~2681、2582~2682、2583~2683、2584~2684、2585~2685、2586~2686、2587~2687、2588~2688、2589~2689、2590~2690、2591~2691、2592~2692、2593~2693、2594~2694、2595~2695、2596~2696、2597~2697、2598~2698、2599~2699、2600~2700、2601~2701、2602~2702、2603~2703、2604~2704、2605~2705、2606~2706、2607~2707、2608~2708、2609~2709、2610~2710、2611~2711、2612~2712、2613~2713、2614~2714、2615~2715、2616~2716、2617~2717、2618~2718、2619~2719、2620~2720、2621~2721、2622~2722、2623~2723、2624~2724、2625~2725、2626~2726、2627~2727、2628~2728、2629~2729、2630~2730、2631~2731、2632~2732、2633~2733、2634~2734、2635~2735、2636~2736、2637~2737、2638~2738、2639~2739、2640~2740、2641~2741、2642~2742、2643~2743、2644~2744、2645~2745、2646~2746、2647~2747、2648~2748、2649~2749、2650~2750、2651~2751、2652~2752、2653~2753、2654~2754、2655~2755、2656~2756、2657~2757、2658~2758、2659~2759、2660~2760、2661~2761、2662~2762、2663~2763、2664~2764、2665~2765、2666~2766、2667~2767、2668~2768、2669~2769、2670~2770、2671~2771、2672~2772、2673~2773、2674~2774、2675~2775、2676~2776、2677~2777、2678~2778、2679~2779、2680~2780、2681~2781、2682~2782、2683~2783、2684~2784、2685~2785、2686~2786、2687~2787、2688~2788、2689~2789、2690~2790、2691~2791、2692~2792、2693~2793、2694~2794、2695~2795、2696~2796、2697~2797、2698~2798、2699~2799、2700~2800、2701~2801、2702~2802、2703~2803、2704~2804、2705~2805、2706~2806、2707~2807、2708~2808、2709~2809、2710~2810、2711~2811、2712~2812、2713~2813、2714~2814、2715~2815、2716~2816、2717~2817、2718~2818、2719~2819、2720~2820、2721~2821、2722~2822、2723~2823、2724~2824、2725~2825、2726~2826、2727~2827、2728~2828、2729~2829、2730~2830、2731~2831、2732~2832、2733~2833、2734~2834、2735~2835、2736~2836、2737~2837、2738~2838、2739~2839、2740~2840、2741~2841、2742~2842、2743~2843、2744~2844、2745~2845、2746~2846、2747~2847、2748~2848、2749~2849、2750~2850、2751~2851、2752~2852、2753~2853、2754~2854、2755~2855、2756、 ~2856、2757~2857、2758~2858、2759~2859、2760~2860、2761~2861、2762~2862、2763~2863、2764~2864、2765~2865、2766~2866、2767~2867、2768~2868、2769~2869、2770~2870、2771~2871、2772~2872、2773~2873、2774~2874、2775~2875、2776~2876、2777~2877、2778~2878、2779~2879、2780~2880、2781~2881、2782~2882、2783~2883、2784~2884、2785~2885、2786~2886、2787~2887、2788~2888、2789~2889、2790~2890、2791~2891、2792~2892、2793~2893、2794~2894、2795~2895、2796~2896、2797~2897、2798~2898、2799~2899、2800~2900、2801~2901、2802~2902、2803~2903、2804~2904、2805~2905、2806~2906、2807~2907、2808~2908、2809~2909、2810~2910、2811~2911、2812~2912、2813~2913、2814~2914、2815~2915、2816~2916、2817~2917、2818~2918、2819~2919、2820~2920、2821~2921、2822~2922、2823~2923、2824~2924、2825~2925、2826~2926、2827~2927、2828~2928、2829~2929、2830~2930、2831~2931、2832~2932、2833~2933、2834~2934、2835~2935、2836~2936、2837~2937、2838~2938、2839~2939、2840~2940、2841~2941、2842~2942、2843~2943、2844~2944、2845~2945、2846~2946、2847~2947、2848~2948、2849~2949、2850~2950、2851~2951、2852~2952、2853~2953、2854~2954、2855~2955、2856~2956, 2857~2957, 2858~2958, 2859~2959, 2860~2960, 2861~2961, 2862~2962, 2863~2963, 2864~2964, 2865~2965, 2866~2966, 2867~2967, 2868~2968, 2869~2969, 2870~2970, 2871~2971, 2872~2972, 2873~2973, 2874~2974, 2875~2975, 2876~2976 , 2877~2977, 2878~2978, 2879~2979, 2880~2980, 2881~2981, 2882~2982, 2883~2983, 2884~2984, 2885~2985, 2886~2986, 2887~2987, 2888~2988, 2889~2989, 2890~2990, 2891~2991, 2892~2992, 2893~2993, 2894~2994, 2895~2995, 2896~2996, 2897~299 7, 2898~2998, 2899~2999, 2900~3000, 2901~3001, 2902~3002, 2903~3003, 2904~3004, 2905~3005, 2906~3006, 2907~3007, 2908~3008, 2909~3009, 2910~3010, 2911~3011, 2912~3012, 2913~3013, 2914~3014, 2915~3015, 2916~3016, 2917~3017, 2918~30 18, 2919-3019, 2920-3020, 2921-3021, 2922-3022, 2923-3023, 2924-3024, 2925-3025, 2926-3026, 2927-3027, or 2928-3028.

[0028] In some embodiments, the nucleic acid stuffer sequence is selected from the group consisting of bases 1-100, 2-101, 3-102, 4-103, 5-104, 6-105, 7-106, 8-107, 9-108, 10-109, 11-110, 12-111, 13-112, 14-113, 15-114, 16-115, 17-116, 18-117, 19-118, 20-119, 21-120, 22-121, 23-122, 24-123, 25-124, 26-125, 27-126, 28-127, 29-128, 30-129, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 44-44, 45-45, 46-46, 47-47, 48-48, 49-50, 51-52, 53-54, 55-56, 57-58, 59-60, 61-62, 63-64, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70, 70-71, 72-72, 73-74, 75-76, 77-78, 78-79, 7 9, 31-130, 32-131, 33-132, 34-133, 35-134, 36-135, 37-136, 38-137, 39-138, 40-139, 41-140, 42-141, 43-142, 44-143, 45-144, 46-145, 47-146, 48-1 47, 49-148, 50-149, 51-150, 52-151, 53-152, 54-153, 55-154, 56-155, 57-156, 58-157, 59-158, 60-159, 61-160, 62-161, 63-162, 64-163, 65-164, 66- 165, 67-166, 68-167, 69-168, 70-169, 71-170, 72-171, 73-172, 74-173, 75-174, 76-175, 77-176, 78-177, 79-178, 80-179, 81-180, 82-181, 83-182, 8 4~183, 85~184, 86~185, 87~186, 88~187, 89~188, 90~189, 91~190, 92~191, 93~192, 94~193, 95~194, 96~195, 97~196, 98~197, 99~198, 100~199, 101~20 0, 102-201, 103-202, 104-203, 105-204, 106-205, 107-206, 108-207, 109-208, 110-209, 111-210, 112-211, 113-212, 114-213, 115-214, 116-215, 117 ~216, 118~217, 119~218, 120~219, 121~220, 122~221, 123~222, 124~223, 125~224, 126~225, 127~226, 128~227, 129~228, 130~229, 131~230, 132~231,133~232、134~233、135~234、136~235、137~236、138~237、139~238、140~239、141~240、142~241、143~242、144~243、145~244、146~245、147~246、148~247、149~248、150~249、151~250、152~251、153~252、154~253、155~254、156~255、157~256、158~257、159~258、160~259、161~260、162~261、163~262、164~263、165~264、166~265、167~266、168~267、169~268、170~269、171~270、172~271、173~272、174~273、175~274、176~275、177~276、178~277、179~278、180~279、181~280、182~281、183~282、184~283、185~284、186~285、187~286、188~287、189~288、190~289、191~290、192~291、193~292、194~293、195~294、196~295、197~296、198~297、199~298、200~299、201~300、202~301、203~302、204~303、205~304、206~305、207~306、208~307、209~308、210~309、211~310、212~311、213~312、214~313、215~314、216~315、217~316、218~317、219~318、220~319、221~320、222~321、223~322、224~323、225~324、226~325、227~326、228~327、229~328、230~329、231~330、232~331、233~332、234~333、235~334、236~335、237~336、238~337、239~338、240~339、241~340、242~341、243~342、244~343、245~344、246~345、247~346、248~347、249~348、250~349、251~350、252~351、253~352、254~353、255~354、256~355、257~356、258-357, 259-358, 260-359, 261-360, 262-361, 263-362, 264-363, 265-364, 266-365, 267-366, 268-367, 269-368, 270-369, 271-370, 272-371, 273-372, 274-373, 275-374, 276-375, 2 77-376, 278-377, 279-378, 280-379, 281-380, or 282-381, which are about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 77-376, 278-377, 279-378, 280-379, 281-380, or 282-381.

[0029] B. Transfer Plasmids In some embodiments, the present specification describes an adeno-associated virus (AAV) plasmid that comprises the nucleic acid stuffer sequence described herein.In certain embodiments, the AAV plasmid comprises an expression cassette that encodes therapeutic peptide, regulatory region, or vector backbone, or a combination thereof.In some embodiments, the rAAV nucleic acid vector comprises a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector, such as a single-stranded or self-complementary recombinant viral genome.

[0030] In some embodiments, the AAV plasmid comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32. In some embodiments, the AAV plasmid comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In some embodiments, the AAV plasmid comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33.

[0031] In some embodiments, the AAV plasmid does not contain a polyG / C sequence. In some embodiments, the AAV plasmid does not contain a sequence with more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive guanine bases. In some embodiments, the AAV plasmid does not contain a sequence with more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive guanine bases within at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 or more nucleobases of the ITR.

[0032] In some embodiments, AAV plasmid comprises an expression cassette as described herein, at least one ITR, and a backbone.In some embodiments, AAV plasmid comprises, in order, a first ITR, an expression cassette, a second ITR, and a backbone.In some embodiments, AAV plasmid comprises an ITR, a promoter, a splice donor / splice acceptor site, a therapeutic peptide, a polyA sequence, an ITR, a first stuffer sequence, an origin of replication, a selection marker, and a second stuffer sequence. In some embodiments, the first stuffer sequence, the second stuffer sequence, or both the first and second stuffer sequences do not contain a CpG island; the first stuffer sequence, the second stuffer sequence, or both the first and second stuffer sequences do not contain more than four consecutive nucleobases of the same identity; the first stuffer sequence, the second stuffer sequence, or both the first and second stuffer sequences contain a GC content of about 40% to about 50%; the first stuffer sequence, the second stuffer sequence, or both the first and second stuffer sequences do not encode an open reading frame (ORF) larger than 20 amino acids; and / or the first stuffer sequence, the second stuffer sequence, or both the first and second stuffer sequences do not contain a restriction enzyme cleavage site.

[0033] Expression cassette In some embodiments, the AAV plasmid comprises a heterologous sequence located between two inverted terminal repeat (ITR) sequences. In some embodiments, the AAV plasmid comprises an expression cassette comprising a heterologous sequence located between two ITR sequences. In some embodiments, the expression cassette is located between the L-ITR (left ITR) and the R-ITR (right ITR). In some embodiments, the expression cassette comprises, in 5' to 3' order, the L-ITR, the heterologous sequence, and the R-ITR.

[0034] In some embodiments, the expression cassette has a length of about 3000 to about 6000 nucleobases. In some embodiments, the expression cassette has a length of about 4000 to about 5000 nucleobases. In some embodiments, the expression cassette has a length of about 4500 to about 5000 nucleobases. In some embodiments, the expression cassette has a length of about 2000 to about 3000 nucleobases. In some embodiments, a vector is provided that is self-complementary and includes a stuffer sequence such that the expression cassette of the vector has a length of about 2000 to about 3000 nucleobases.

[0035] In some embodiments, the expression cassette comprises a stuffer sequence. In some embodiments, the stuffer sequence has a length of about 1000 to about 10000, about 1000 to about 9000, about 1000 to about 8000, about 1000 to about 7000, about 1000 to about 6000, about 1000 to about 5000, about 1000 to about 4000, about 1000 to about 3000, or about 1000 to about 2000 nucleobases. In some embodiments, the second stuffer sequence has a length of about 2000 to about 3000 nucleobases. In some embodiments, the stuffer sequence has a length of 2235 nucleobases. In some embodiments, the second stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous base pair sequences of SEQ ID NO: 7. In some embodiments, the stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a SEQ ID NO: 7.

[0036] In some embodiments, the expression cassette comprises a therapeutic peptide. In some embodiments, the expression cassette comprises at least one regulatory region. In some embodiments, a stuffer sequence is located between the therapeutic peptide and the regulatory region. In some embodiments, the therapeutic peptide is located between the stuffer sequence and the R-ITR. In some embodiments, the stuffer sequence is located between the therapeutic peptide and the L-ITR.

[0037] Therapeutic peptides In some embodiments, the AAV plasmids disclosed herein are used to encode diagnostic or therapeutic proteins, polypeptides, or molecular markers, The present invention includes sequences encoding biologically active fragments of photosensitive opsins, adrenergic agonists, anti-apoptotic factors, apoptosis inhibitors, cytokine receptors, cytokines, cytotoxins, erythropoietic agents, glutamic acid decarboxylase, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, kinases, kinase inhibitors, nerve growth factors, netrins, neuroactive peptides, neuroactive peptide receptors, neurofactors, neurofactor receptors, neuropilins, neurotrophic factors, neurotrophins, neurotrophin receptors, N-methyl-D-aspartate antagonists, plexins, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, proteolytic proteins, proteolytic protein inhibitors, semaphorins, semaphorin receptors, serotonin transport proteins, serotonin uptake inhibitors, serotonin receptors, serpins, serpin receptors, tumor suppressors, or any combination thereof. In some embodiments, the photosensitive opsin comprises rhodopsin, melanopsin, cone opsin, channelrhodopsin, or bacterial, archaeal related opsin, a biologically active fragment of any of these, or a combination thereof.

[0038] In some embodiments, the AAV plasmids described herein comprise a sequence encoding a therapeutic peptide or a biologically active fragment thereof selected from the group consisting of GUCY2D, RS1, CNBG3, ADAMTS10, ABCA4, or frataxin. In some embodiments, the AAV plasmids encode the polypeptides RPE65, bestrophin (BEST1), REP1, MERTK, SOD2, MYO6A, MFRP, LRAT, KCNJ13, ornithine aminotransferase (OAT), CNTF, GDNF, BDNF, IL6, LIF, XIAP, STATS, nactallopin (nyx), metabotropic glutamate receptor 6-mGluR6 (Grm6), transient receptor potential melastatin 1 (TRPM1), G protein-coupled receptor 179 (GPR179), and the G proteins, Gβ5, βP3, Gα0. 1 / 2 , Gγ13, RGS7, RGS11, REAP, MYO7A, OPN1MW, OPN1LW, CNGA3, CNGB1, Rho, PDE6b, PDE6a, GNAT2, PDE6c, RPGR, RPGR-ORF15, RPGRIP, CLRN1, Ush1c / Harmonin, Mt-ND4, P1-ND4, CYP4V2, any combination or peptide fragment thereof. In some embodiments, the AAV plasmids described herein comprise sequences encoding a CRISPR-Cas system. In some embodiments, the AAV plasmids described herein comprise sequences encoding a CRISPR-Cas9 system.

[0039] In some embodiments, the sequence encoding the therapeutic peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous base pair sequences of SEQ ID NO: 4. In some embodiments, the sequence encodes a therapeutic peptide that comprises a sequence that is nearly identical or at least nearly identical to at least 50 contiguous amino acids of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the peptide comprises SEQ ID NO: 4. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34. In some embodiments, the peptide comprises SEQ ID NO: 34. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35. In some embodiments, the peptide comprises SEQ ID NO: 35. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36. In some embodiments, the peptide comprises SEQ ID NO: 36.In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments, the peptide comprises SEQ ID NO: 37. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38. In some embodiments, the peptide comprises SEQ ID NO: 38. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. In some embodiments, the peptide comprises SEQ ID NO: 39. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40. In some embodiments, the peptide comprises SEQ ID NO: 40. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In some embodiments, the peptide comprises SEQ ID NO: 41. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42. In some embodiments, the peptide comprises SEQ ID NO: 42.In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43. In some embodiments, the peptide comprises SEQ ID NO: 43. In some embodiments, the peptide comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44. In some embodiments, the peptide comprises SEQ ID NO: 44.

[0040] b. Regulatory region Any of the vector systems of the present disclosure may contain regulatory elements that are functional in the host cell in which the vector is intended to be expressed. Regulatory elements include, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.

[0041] Any of the vector systems of the present disclosure may include a promoter sequence operably linked to a nucleotide sequence encoding a desired polypeptide.Promoters contemplated for use in the present disclosure include, but are not limited to, cytomegalovirus (CMV) promoter, SV40 promoter, promoter derived from human myosin 7a gene, Rous sarcoma virus (RSV) promoter, chimeric CMV / chicken β-actin promoter (CBA) and truncated form of CBA (smCBA) (see, e.g., Haire et al. 2006 and U.S. Pat. No. 8,298,818). Additional photoreceptor-specific human rhodopsin kinase (hGRK1) promoters, synapsin promoters, glial fibrillary acidic protein (GFAP) promoters, rod-specific IRBP promoters, VMD2 (vitreous macular dystrophy / Best's disease) promoters, RPE-specific vitreous macular dystrophy 2 [VMD2] promoters, EF1-alpha promoter sequences, PGK promoters, Pleiades "PleXXX" promoters, red / green cone opsin promoters PR2.1 and PR1.7 are also contemplated as being useful in implementing various aspects of the present disclosure. Exemplary photoreceptor cell-specific promoters include, but are not limited to, hGRK1, IRBP, rod opsin, NRL, GNAT2e-IRBP, L / M opsin, and cone arrestin promoters. In some embodiments, the promoter comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2.

[0042] In certain embodiments, the promoter is a chimeric CMV-β actin promoter. In certain embodiments, the promoter is a tissue-specific promoter that exhibits selective activity in one or a group of tissues, but has low or no activity in other tissues. In certain embodiments, the promoter is a photoreceptor-specific promoter. In further embodiments, the promoter is preferably a cone cell-specific promoter or a rod cell-specific promoter, or any combination thereof. In certain embodiments, the promoter is a promoter of the human MYO7A gene. In further embodiments, the promoter comprises a promoter derived from the cone transducin alpha (TαC) gene. In certain embodiments, the promoter is a promoter derived from the human GNAT2. Other promoters contemplated within the scope of the present disclosure include, but are not limited to, rhodopsin promoter (human or mouse), cGMP-phosphodiesterase β subunit promoter, retinitis pigmentosa-specific promoter, RPE cell-specific promoter [e.g., vitreous macular dystrophy 2 (VMD2) promoter (Best1) (Esumi et al., 2004)], or any combination thereof.

[0043] The promoter can be incorporated into the vector using standard techniques known to those skilled in the art of molecular biology and / or virology.Multiple copies of the promoter and / or multiple separate promoters can be used in the vector of the present disclosure.In one such embodiment, the promoter can be positioned at approximately the same distance from the transcription start site as it is from the transcription start site in its natural genetic environment, although of course some variation in this distance is tolerated without substantial reduction in promoter activity.In carrying out the present disclosure, one or more transcription start sites are typically included in the vector disclosed.

[0044] The vector of the present disclosure may further comprise one or more transcription termination sequences, one or more translation termination sequences, one or more signal peptide sequences, one or more internal ribosome entry sites (IRES), and / or one or more enhancer elements, or any combination thereof. The transcription termination region can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. The transcription termination sequence may be located downstream of the coding sequence to provide efficient termination. In some embodiments, the vector comprises a self-cleaving peptide. In some embodiments, the self-cleaving peptide allows for multiple simultaneous expression. In some embodiments, the self-cleaving peptide comprises a 2A self-cleaving peptide. In some embodiments, the peptide comprises P2A, E2A, F2A, or T2A.

[0045] Any of the disclosed polynucleotide vectors may further comprise one or more post-transcriptional regulatory sequences or one or more polyadenylation signals, including, but not limited to, a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WRPE), a polyadenylation signal sequence, or an intron / exon junction / splicing signal, or any combination thereof. In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). In some embodiments, the WPRE comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5. In some embodiments, the expression cassette comprises a pGH polyA sequence. In some embodiments, the pGH polyA sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6. In some embodiments, the intron / exon junction / splicing signal comprises SV40SD / SA. In some embodiments, the SV40SD / SA comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:34.

[0046] Signal peptide sequences are amino-terminal peptide sequences that encode information critical for the location of a polypeptide that is operably linked to one or more post-translational cellular destinations, including, for example, specific organelle compartments or sites of protein synthesis and / or activity, as well as the extracellular environment.

[0047] Enhancer (cis-acting regulatory element that increases gene transcription) can also be included in one of the disclosed AAV-based vector systems. A variety of enhancer elements are known to those skilled in the art, including but not limited to CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, SV40 enhancer element, and combinations and / or derivatives thereof. One or more nucleic acid sequences that direct or regulate the polyadenylation of the mRNA encoded by the structural gene of interest can also be optionally included in one or more of the vectors of the present disclosure.

[0048] backbone In some embodiments, the AAV plasmid comprises a backbone. In some embodiments, the backbone comprises a region of the plasmid outside the ITR. In some embodiments, the stuffer sequence is located outside the expression cassette. In some embodiments, the backbone sequence is at least about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, or about 10000 nucleobases. In some embodiments, the backbone sequence is about 4000 to about 10000, about 4000 to about 9000, about 4000 to about 8000, about 4000 to about 7000, about 4000 to about 6000, or about 4000 to about 5000 nucleobases. In some embodiments, the backbone sequence is about 5000 to about 10000, about 5000 to about 9000, about 5000 to about 8000, about 5000 to about 7000, or about 5000 to about 6000 nucleobases. In some embodiments, the backbone sequence is about 1000 to about 8000, about 2000 to about 8000, about 3000 to about 8000, about 4000 to about 8000, about 500 to about 8000, or about 6000 to about 8000 nucleobases. In some embodiments, the backbone sequence is sufficiently large to prevent reverse packaging into AAV particles.

[0049] In some embodiments, the backbone comprises an origin of replication, and in some embodiments, the nucleic acid stuffer sequence is located 3' to the origin of replication. In some embodiments, the nucleic acid stuffer sequence is located between the origin of replication and the ITR. In some embodiments, the nucleic acid stuffer sequence is located such that the ITR is about 2000 to about 4000 nucleobases away from the origin of replication. In some embodiments, the nucleic acid stuffer sequence is located such that the ITR is about 1000 to about 4000 nucleobases away from the origin of replication. In some embodiments, the nucleic acid stuffer sequence is located such that the ITR is about 3000 to about 3500 nucleobases away from the origin of replication. In some embodiments, the nucleic acid stuffer sequence is located such that the ITR is about 500, 1000, 2000, 3000, 4000, or more than 5000 nucleobases away from the origin of replication. In some embodiments, the nucleic acid stuffer sequence is positioned such that the ITRs are less than about 500, 1000, 2000, 3000, 4000, or 5000 nucleobases away from the origin of replication. In some embodiments, the stuffer sequence is positioned such that the ITRs are less than about 3124 nucleobases away from the origin of replication.

[0050] In some embodiments, the backbone sequence comprises two stuffer sequences. In some embodiments, the first stuffer sequence is located between the R-ITR and the origin of replication. In some embodiments, the first stuffer sequence is located between the R-ITR and the antibiotic resistance gene. In some embodiments, the second stuffer sequence is located between the origin of replication and the L-ITR. In some embodiments, the second stuffer sequence is located between the antibiotic resistance gene and the L-ITR. In some embodiments, the backbone comprises, in order from 5' to 3', a first stuffer sequence, an origin of replication, an antibiotic resistance gene, and a second stuffer sequence. In some embodiments, the backbone comprises, in order from 3' to 5', a first stuffer sequence, an origin of replication, an antibiotic resistance gene, and a second stuffer sequence.

[0051] In some embodiments, the first stuffer sequence has a length of about 1000 to about 10000, about 2000 to about 9000, about 3000 to about 8000, about 4000 to about 6000 nucleobases. In some embodiments, the first stuffer sequence has a length of about 3000 to about 3500 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 3028 nucleobases. In some embodiments, the first stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous base pair sequences of SEQ ID NO:8. In some embodiments, the first stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8.

[0052] In some embodiments, the second stuffer sequence has a length of about 100 to about 1000, about 100 to about 900, about 100 to about 800, about 100 to about 700, about 100 to about 600, about 100 to about 500, about 100 to about 400, about 100 to about 300, or about 100 to about 200 nucleobases. In some embodiments, the second stuffer sequence has a length of about 100 to about 500 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 381 nucleobases. In some embodiments, the second stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous base pair sequences of SEQ ID NO: 11. In some embodiments, the second stuffer sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11.

[0053] In some embodiments, the AAV plasmid comprises a selection marker. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the AAV plasmid comprises a kanamycin resistance gene. In some embodiments, the AAV plasmid does not comprise an antibiotic gene. In some embodiments, the AAV plasmid does not comprise an ampicillin antibiotic resistance gene.

[0054] In some embodiments, the selection marker is a non-antibiotic selection system. In some embodiments, the non-antibiotic selection system comprises an RNA-OUT sequence and an R6K sequence.

[0055] In some embodiments, the backbone has no more than one origin of replication, hi some embodiments, the backbone does not have an M13 origin of replication.

[0056] ITR In some embodiments, the plasmids described herein include at least one sequence that includes an inverted repeat (ITR). The ITR sequence can be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or from multiple serotypes. In some embodiments, the ITR sequence is derived from AAV2 or AAV6. In some embodiments, the ITR sequence of the first serotype is derived from AAV3, AAV2, or AAV6. In other embodiments, the ITR sequence of the first serotype is derived from AAV1, AAV5, AAV8, AAV9, or AAV10. In some embodiments, the ITR sequence is of the same serotype as the capsid (e.g., AAV3 ITR sequence and AAV3 capsid, etc.).

[0057] ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, Pa.; Cellbiolabs, San Diego, Calif.; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, Mass., and gene delivery to skeletal muscle has resulted in sustained expression and systemic delivery of therapeutic proteins). In some embodiments, the nucleic acid vector comprises a pTR-UF-11 plasmid backbone, which is a plasmid containing AAV2 ITRs. This plasmid is commercially available from the American Type Culture Collection (ATCC MBA-331).

[0058] In some embodiments, the ITR sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the ITR sequence comprises a sequence that is about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:51.

[0059] C. Triple transfection system In certain aspects, a triple plasmid transfection system for the production of AAV plasmids is described herein. In some embodiments, the triple plasmid transfection system comprises an AAV vector plasmid, a rep / cap plasmid, and a helper plasmid as described herein.

[0060] In one aspect, a double plasmid transfection system for the production of AAV plasmid is described herein. In some embodiments, the double plasmid transfection system comprises an AAV vector plasmid (also called a transfer plasmid) as described herein and a helper plasmid, wherein the helper plasmid comprises a rep / cap sequence.

[0061] Simply by way of example, the same AAV plasmid can be produced using either a triple plasmid system or a double plasmid transfection system. In one example, the triple transfection system includes pTR-UF11(CBA-GFP) (SEQ ID NO: 45), helper plasmid pALD-X80 (SEQ ID NO: 47), and rep / cap plasmid pACG2 (SEQ ID NO: 46) transfected into HEK293T cells. In another embodiment, the two-plasmid system consists of pTR-UF11(CBA-GFP) (SEQ ID NO: 45) and pDG (SEQ ID NO: 48). Plasmid pDG is a helper plasmid that also contains elements that drive the expression of AAV2 rep / cap. These three-plasmid and two-plasmid systems result in the same AAV2-GFP product.

[0062] Helper plasmid In some embodiments, the systems described herein include a helper plasmid. In some embodiments, the helper plasmid includes an E1a gene, an E1b gene, an E4 gene, an E2a, an e3 gene, an E5 gene, a Fiber gene, or a VA gene, or a combination thereof. In some embodiments, the helper plasmid includes a mutated Fiber gene. In some embodiments, the Fiber gene includes about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence of SEQ ID NO: 12. In some embodiments, the Fiber gene comprises about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence of SEQ ID NO: 13. In some embodiments, the helper plasmid does not comprise the Fiber gene.

[0063] In some embodiments, the helper plasmid comprises a pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), or pDP8.ape plasmid. In some embodiments, the helper plasmid comprises about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence to SEQ ID NO:47. In some embodiments, the helper plasmid comprises SEQ ID NO:47.

[0064] Rep / Cap Plasmid In some embodiments, the systems described herein include a rep / cap plasmid that includes rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (encoding VP1, VP2, and VP3, including modified VP3 regions described herein). In some embodiments, the rep / cap plasmid is transfected into a producer cell line such that rAAV particles can be packaged and subsequently purified. In some embodiments, the rep / cap plasmid includes about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence to SEQ ID NO:46. In some embodiments, the rep / cap plasmid includes SEQ ID NO:46.

[0065] In some embodiments, the rep gene is the rep gene from AAV2. In some embodiments, the cap gene is from AAV2. In some embodiments, the rep gene is the rep gene from AAV12. In some embodiments, the Rep gene is Rep2-6. In some embodiments, the Rep gene comprises about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence to SEQ ID NO:49. In some embodiments, the Rep gene comprises SEQ ID NO:49.

[0066] In some embodiments, the cap gene is derived from AAV12. In some embodiments, the cap gene comprises modifications to the gene to produce a modified capsid protein as described herein. In some embodiments, the Rep gene comprises Rep78, Rep68, Rep52, or Rep40. In some embodiments, the cap gene comprises VP1, VP2, VP3, or variants thereof. In some embodiments, the cap gene comprises about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence to SEQ ID NO:50. In some embodiments, the cap gene comprises SEQ ID NO:50.

[0067] In some embodiments, the present disclosure provides improved rAAV particles derived from a number of different serotypes, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV44.9(E531D), and combinations thereof. In some embodiments, the capsid protein sequence is set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31. In some embodiments, the capsid protein sequence comprises SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31. In some embodiments, the capsid protein sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

[0068] In some embodiments, the capsid comprises a capsid comprising an unnatural amino acid substitution at an amino acid residue of a wild-type AAV2 capsid set forth in SEQ ID NO: 22. In some embodiments, the unnatural amino acid substitution comprises one or more of Y272F, Y444F, T491V, Y500F, Y700F, Y704F, Y730F, or a combination thereof. In some embodiments, the capsid comprises an unnatural amino acid substitution at an amino acid residue of a wild-type AAV6 capsid set forth in SEQ ID NO: 26. In some embodiments, the unnatural amino acid substitution comprises one or more of Y445F, Y705F, Y731F, T492V, S663V, or a combination thereof.

[0069] In some embodiments, the capsid comprises AAV2G9, a mutant of AAV2.

[0070] In some embodiments, the capsid comprises a non-natural amino acid substitution at amino acid residue 533 of the wild-type AAV8 capsid, as set forth in SEQ ID NO: 28. In some embodiments, the non-natural amino acid substitution is E533K, Y733F, or a combination thereof. In some embodiments, the capsid comprises AAV7BP2, a mutant of AAV8.

[0071] In some embodiments, the capsid comprises a non-natural amino acid substitution of the wild-type AAV2 capsid set forth in SEQ ID NO: 22. In some embodiments, the capsid comprises

[0072] (a) Y444F,

[0073] (b) Y444F+Y500F+Y730F,

[0074] (c) Y272F + Y444F + Y500F + Y730F,

[0075] (d) Y444F+Y500F+Y730F+T491V, or

[0076] (e) Y272F+Y444F+Y500F+Y730F+T491V, or one or more of the corresponding equivalent amino acid positions in any one of the wild-type AAV1, AAV3, AAV4, AAV5, AAV7, AAV9, or AAV10 capsid proteins as set forth in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:30, respectively.

[0077] In some embodiments, the capsid comprises a non-natural amino acid substitution of the wild-type AAV6 capsid set forth in SEQ ID NO: 26. In some embodiments, the capsid comprises

[0078] (a) Y445F,

[0079] (b) Y705F+Y731F,

[0080] (c) T492V,

[0081] (d) Y705F+Y731F+T492V,

[0082] (e) S663V, or

[0083] (f) S663V+T492V Contains one or more of the following:

[0084] In various embodiments, the rAAV particles comprise one of the following capsids: capsid mutants of AAV2, DGE-DF (also known as "V1V4 VR-V"), P2-V2, P2-V3, P2-V1 (also known as ME-B), and ME-B(Y-F+TV). The DGE-DF capsid mutant contains aspartic acid, glycine, glutamic acid, aspartic acid, and phenylalanine at amino acid positions 492, 493, 494, 499, and 500 of wild-type AAV2 VP1. The P2-V2 capsid mutant contains alanine, threonine, proline, aspartic acid, phenylalanine, and aspartic acid at positions 263, 490, 492, 499, 500, and 530 of AAV2 VP1. The P2-V3 capsid mutant contains asparagine, alanine, phenylalanine, alanine, asparagine, valine, threonine, arginine, aspartic acid, and aspartic acid at AAV2 VP1 positions 263, 264, 444, 451, 454, 455, 459, 527, 530, and 531. The ME-B(Y-F+TV) capsid mutant contains aspartic acid, glycine, glutamic acid, aspartic acid, and phenylalanine at positions 492, 493, 494, 499, and 500, respectively, of AAV2 VP1 (SEQ ID NO:22), SAAGADXAXDS (SEQ ID NO:52) at positions 546-556 of AAV2 VP1, and the following substitutions, Y272F, Y444F, and T491V.

[0085] In other embodiments, the rAAV particles comprise a capsid selected from AAV6(3pMut), AAV2(quadYF+TV), or AAV2(trpYF). In some embodiments, the rAAV particles comprise any of the capsid mutants described in International Publication WO2018 / 156654.

[0086] In some embodiments, the AAV particles comprise capsids including a DGE-DF capsid, a P2-V2 capsid, a P2-V3 capsid, or a ME-B(Y-F+TV) capsid for enhanced transduction of the rAAV particles in retinal cells. In some embodiments, the AAV particle comprises a capsid selected from AAV2(Y444F), AAV2(Y444F+Y500F+Y730F), AAV2(Y272F+Y444F+Y500F+Y730F), AAV2(Y444F+Y500F+Y730F+T491V) and AAV2(Y272F+Y444F+Y500F+Y730F+T491V), AAV6(Y445F), AAV6(Y705F+Y731F), AAV6(Y705F+Y731F+T492V), AAV6(S663V), AAV6(T492V), or AAV6(S663V+T492V).

[0087] In some embodiments, an rAAV polynucleotide or nucleic acid vector of the disclosure may be contained within a virion having a serotype selected from the group consisting of AAV serotype 1, AAV serotype 2, AAV serotype 3, AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, or AAV serotype 10, AAV449.5(E531D), or any other serotype known to one of skill in the art of viruses.

[0088] Production In some embodiments, methods of rAAV particle production are described herein. In some embodiments, one or more helper plasmids are produced or obtained. In some embodiments, a helper plasmid and a rep / cap plasmid are produced or obtained. In some embodiments, the one or more helper plasmids contain the rep and cap ORFs for the desired AAV serotype and the adenovirus VA, E2A (DBP), and E4 genes. In some embodiments, the rep and cap ORFs for the desired AAV serotype and the adenovirus VA, E2A (DBP), and E4 genes are under the transcriptional control of their native promoters. In some embodiments, the cap ORF contains one or more modifications to generate modified capsid proteins as described herein. In some embodiments, HEK293 cells (available from ATCC®) are transfected with a plasmid containing a helper plasmid and a nucleic acid vector as described herein via CaPO4-mediated transfection, lipids, or polymer molecules such as polyethyleneimine (PEI). In some embodiments, the HEK293 cells are incubated for at least about 60 hours to allow for rAAV particle production. In some embodiments, the cells are then incubated for at least 60 hours to allow for rAAV particle production. In some embodiments, the rAAV particles are purified. In some embodiments, the rAAV particles are purified by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

[0089] II. Method In some aspects, the present disclosure provides a method for treating or ameliorating a disease or condition, such as an eye disease, in a human or animal using gene therapy and the AAV-based dual vector system of the present disclosure. In certain embodiments, the method of the present disclosure comprises administering a vector system of the present disclosure that encodes a polypeptide that contributes to the treatment or amelioration of the disease or condition. In certain embodiments, the vector of the present disclosure is provided in an AAV virus or virion. The vector system can be administered in vivo or ex vivo.

[0090] In some embodiments, the rAAV vector constructs disclosed herein may be administered via intravitreal injection, subretinal injection, oral, parenteral, intraocular, intravenous, intranasal, intraarticular, intramuscular, subcutaneous, subILM (wherein the vector is placed between the internal limiting membrane and the retina), or a combination thereof. In some embodiments, the rAAV vector constructs disclosed herein are administered to the subject once. In some embodiments, the rAAV vector constructs are administered to the subject for one or more administration periods, for example, at least once daily, twice daily, three times daily, once weekly, twice weekly, once monthly, twice monthly, or at least once yearly. In some embodiments, the AAV vector-based therapeutic may be provided continuously, one or more times at intervals every week, every month, or less frequently, as may be necessary to achieve treatment or improvement of one or more symptoms of the disease or disorder being treated. In some embodiments, the pharmaceutical compositions disclosed herein may be administered one or more times, for example, daily, weekly, biweekly, or monthly, hourly, or upon recurrence, relapse, or progression of the disease, disorder, or condition being treated.

[0091] In some embodiments, the vector system is administered to hair cells of the ear, for example, by injection into the utricle, one of two gravity-sensitive saccular otolith organs, as described in Lee et al., Hearing Research Vol. 394 (2020) 107882 (herein incorporated by reference). For example, administration to hair cells of the ear can be by round window injection or during cochlear implant surgery. In certain embodiments, the vector system of the present disclosure is administered to humans or animals by intraocular, intravitreal, or subretinal injection.

[0092] In some embodiments, administration of any of the disclosed vectors, virions, or compositions to a subject in need results in partial or complete restoration of melanosome migration in retinal pigment epithelial (RPE) cells. In exemplary embodiments, administration of any of the polynucleotide vector systems, virions, or compositions results in partial or complete restoration of vision loss.

[0093] In some embodiments, methods of use of the described rAAV particles or vectors, virions, expression systems, compositions, and host cells are described herein in the preparation of a medicament for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, dysfunction, disorder, abnormal condition, defect, injury, or trauma in an animal, particularly in the eye. In some embodiments, the method comprises administering one or more of the described vectors, virions, viral particles, host cells, compositions, or a plurality thereof directly to the vitreous of one or both eyes of a mammal in need thereof in an amount and for a time sufficient to diagnose, prevent, treat, or ameliorate one or more symptoms of such disease, dysfunction, disorder, abnormal condition, defect, injury, or trauma in one or both eyes of the affected animal.

[0094] In some aspects, the disclosure provides methods of use of the particles, vectors, virions, expression systems, compositions, and host cells described herein in methods for treating or ameliorating symptoms of various defects in the mammalian eye, particularly one or more defects in human photoreceptor or RPE cells, or in the preparation of a medicament for treating or ameliorating symptoms. In some embodiments, ocular diseases and disorders (e.g., caused by one or more genetic defects in PR or RPE cells) for which symptoms are treated or ameliorated include retinitis pigmentosa, Leber's congenital amaurosis (e.g., LCA10), age-related macular degeneration (AMD), wet AMD, dry AMD, uveitis, Best's disease, Stargardt's disease, Usher syndrome, geographic atrophy, diabetic retinopathy, retinopathy, achromatosis, corredoremia, Bardet-Beer syndrome, autosomal dominant umbilical muscular dystrophy (CORD6), glaucoma, including primary open-angle glaucoma, Freidrich's ataxia, glycogen storage disease (ocular manifestation), congenital stationary night blindness, Leber's hereditary optic neuropathy (LHON), or Vitti crystalline dystrophy. In some embodiments, the methods include intravitreal or subretinal administration of one or more of the described particle vectors, virions, host cells, or compositions to one or both eyes of a subject in need thereof in an amount and for a time sufficient to treat or ameliorate symptoms of such a defect in an affected mammal. In some embodiments, the methods include prophylactic treatment of animals suspected of having such a disease, or administration of such compositions to animals at risk of developing such a disease, after diagnosis or prior to the onset of symptoms. In some embodiments, the rAAV particles are not included in a chimeric viral / non-viral nanoparticle.

[0095] III. Pharmaceutical Compositions Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be, for example, a solvent or liquid dispersion medium, including water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The appropriate fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar, buffer solution, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0096] The present disclosure also provides a pharmaceutical composition comprising the vector system of the present disclosure in combination with a pharma- ceutically acceptable carrier. A pharmaceutical composition comprising a quantity of the compound and adapted for topical or parenteral administration constitutes a preferred embodiment of the present disclosure. The dose administered to a patient, particularly a human, in the context of the present disclosure should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and preferably without causing side effects or morbidity at or below an acceptable level. Those skilled in the art will recognize that the dosage depends on a variety of factors, including the subject's condition (health), the subject's weight, the type of concomitant treatment (if any), the frequency of treatment, the therapeutic ratio, and the severity and stage of the pathological condition.

[0097] The present disclosure also provides a kit that includes the vector system of the present disclosure in one or more containers.The kit of the present disclosure can optionally include a pharma- ceutically acceptable carrier and / or diluent.In certain embodiments, the kit of the present disclosure includes one or more other components, adjuvants, or adjuvants as described herein.In certain embodiments, the kit of the present disclosure includes instructions or packaging that describe how to administer the vector system contained in the kit to a selected mammalian recipient.

[0098] The container of the disclosed kit may be any suitable material, such as glass, plastic, metal, etc., and any suitable size, shape, or configuration. In certain embodiments, the vector system of the present disclosure is provided in the kit as a solid. In another embodiment, the vector system of the present disclosure is provided in the kit as a liquid or solution. In some embodiments, the kit may include one or more ampoules or syringes that contain the vector system of the present disclosure in a suitable liquid or solution form.

[0099] Further contemplated herein are kits containing premixes of any of the disclosed dual vectors (first and second vectors). These premixes may be in a single container and / or a single drug product in suitable liquid or solution form.

[0100] The present disclosure also provides for the use of the buffers and compositions of the present disclosure in the manufacture of a medicament for treating, preventing, or ameliorating a symptom of a disease, disorder, dysfunction, injury, or trauma, including, but not limited to, the treatment, prevention, and / or prophylaxis of a disease, disorder, or dysfunction, and the amelioration of one or more symptoms of such disease, disorder, or dysfunction.

[0101] The amount of AAV composition and the administration time of such composition are within the purview of a person skilled in the art who has the benefit of the teachings of the present invention.The administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, for example, a single injection of a sufficient number of infectious particles to provide a therapeutic benefit to the patient undergoing such treatment.Alternatively, in some situations, it may be desirable to provide multiple or continuous administrations of AAV vector composition, either over a relatively short period of time or over a relatively long period of time, as can be determined by the physician supervising the administration of such composition.

[0102] For example, the number of infectious particles administered to a mammal is approximately 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or even higher numbers of infectious particles / mL, and may be given as a single dose (or divided into two or more administrations, etc.) as may be required to achieve treatment of the particular disease or disorder being treated. Indeed, in some embodiments, it may be desirable to administer two or more different rAAV particle or vector-based compositions, either alone or in combination with one or more other diagnostic agents, drugs, bioactive agents, etc., to achieve the desired effect of a particular regimen or treatment.

[0103] To express a therapeutic agent according to the present disclosure, rAAV particles may be prepared that contain a nucleic acid segment encoding the therapeutic agent under the control of one or more promoters. To place a sequence "under the control" of a promoter, the 5' end of the transcription initiation site of the transcription reading frame is generally located about 1 to about 50 nucleotides "downstream" (e.g., 3') of the selected promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded polypeptide. This is what is meant by "recombinant expression" in this context. In some embodiments, the recombinant vector construct comprises a capsid-protein modified rAAV vector that contains an RPE cell-specific promoter or a photoreceptor cell-specific promoter operably linked to at least one nucleic acid segment encoding one or more diagnostic and / or therapeutic agents.

[0104] When the use of such vectors is intended for the introduction of one or more exogenous proteins, polypeptides, peptides, ribozymes, and / or antisense oligonucleotides into a particular cell transfected with the vector, the rAAV particles disclosed herein may be employed to deliver one or more exogenous polynucleotides to a selected host cell, for example, to one or more selected cells in a mammalian eye. In some embodiments, the cell is a retinal ganglion cell, a Muller glia, a bipolar cell, an astrocyte, an amacrine cell, a trabecular meshwork cell, a photoreceptor cell, or a retinal pigment epithelial cell. In some embodiments, the cell is a photoreceptor cell. In some embodiments, the cell is a retinal pigment epithelial (RPE) cell.

[0105] In some embodiments, the number of viral particles administered to a subject is 10 6 ~10 14 Particles / ml range or 10 3 ~10 15 In one embodiment, the amount of the 10 13 Higher numbers of viral particles / ml may be administered. In some embodiments, the number of viral particles administered to a subject is greater than 10 6~10 14 Vector genomes (vgs) / ml range or 10 3 ~10 15 In one embodiment, the range may be on the order of 10 vgs / ml. 13 Viral particles are administered in higher numbers than vgs / ml. The viral particles can be administered as a single dose or can be divided into two or more administrations, as may be required to achieve treatment of the particular disease or disorder being treated.

[0106] In some embodiments, the present disclosure provides formulations of one or more virus-based compositions described herein in a pharma- ceutically acceptable solution for administration to a cell or an animal, either alone or in combination with one or more other therapeutic modalities, particularly for the treatment of diseases affecting human cells, tissues, and humans.

[0107] If desired, the rAAV particles described herein can be administered in combination with other agents, such as, for example, proteins or polypeptides or various pharmacologic active agents, including systemic or local administration of one or more therapeutic polypeptides, biologically active fragments, or variants thereof. In fact, there is virtually no limit to the other components that may be included, provided that the additional agent does not cause significant adverse effects upon contact with target cells or host tissues. Thus, the rAAV particles can be delivered with various other agents as required in a particular case. Such compositions may be purified from host cells or other biological sources, or may be chemically synthesized as described herein.

[0108] The formulation of pharma- ceutically acceptable buffers, excipients, and carrier solutions is well known to those of skill in the art, as is the development of appropriate dosages and treatment regimens for use of the particular compositions described herein in various treatment regimens, including, for example, oral, parenteral, intraocular (e.g., subretinal or intravitreal), intravenous, intranasal, intraarticular, intrautricular, intracochlear, and intramuscular administration and formulations.

[0109] Typically, these formulations will contain at least about 0.1% or more of a therapeutic agent (e.g., rAAV particles), although the percentage of active ingredient may of course vary and may conveniently be from about 1 or 2% to about 70% or 80% or more by weight or volume of the total formulation. Of course, the amount of therapeutic agent in each therapeutically useful composition that may be prepared will be such that an appropriate dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and thus various dosages and treatment regimens may be desirable.

[0110] The term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which rAAV particles are administered. Such pharmaceutical excipients can be sterile liquids such as water and oil, including petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or synthetic oil. Physiological saline solution and aqueous dextrose and glycerol solutions can also be used as liquid carriers. Exemplary excipients and vehicles include, but are not limited to, HA, BSS, artificial CSF, PBS, lactated Ringer's solution, TMN200 solution, polysorbate 20, and poloxamer 100.

[0111] The amount of rAAV particle composition and the administration time of such composition are within the purview of a person skilled in the art who has the benefit of the teachings of the present invention.However, administration of a therapeutically effective amount of the disclosed composition is likely to be achieved by a single administration, for example, a single injection of a sufficient number of viral particles to provide a therapeutic benefit to the patient undergoing such treatment.Alternatively, in some situations, it may be desirable to provide multiple or continuous administrations of the composition, either over a relatively short period of time or over a relatively long period of time, as can be determined by the physician supervising the administration of such compositions.

[0112] Exemplary compositions can include rAAV particles or nucleic acid vectors, either alone or in combination with one or more additional active components, which can be obtained from natural or recombinant sources or chemically synthesized.

[0113] IV. Definition Unless otherwise defined, all technical terms, designations, and other technical and scientific terms or terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference to what is commonly understood in the art.

[0114] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered to have all possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0115] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "sample" includes a plurality of samples, mixtures thereof.

[0116] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present (e.g., detecting). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence" can include determining the amount of something present in addition to determining whether it is present or absent, depending on the context.

[0117] The terms "subject", "individual", or "patient" are often used interchangeably herein. A "subject" can be a biological entity that contains expressed genetic material. The biological entity can be, for example, a plant, an animal, or a microorganism, including bacteria, viruses, fungi, and protozoa. A subject can be tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal. A mammal can be a human. A subject can be diagnosed or suspected of being at high risk for a disease. In some cases, a subject is not necessarily diagnosed or suspected of being at high risk for a disease.

[0118] The term "in vivo" is used to describe events that take place inside the body of a subject.

[0119] The term "ex vivo" is used to describe events that occur outside of a subject's body. An ex vivo assay is not performed on a subject. Rather, it is performed on a sample that is separated from a subject. An example of an ex vivo assay that is performed on a sample is an "in vitro" assay.

[0120] The term "in vitro" is used to describe events that occur in a container for holding laboratory reagents such that the material is separated from the biological source from which it is obtained. In vitro assays can include cell-based assays in which live or dead cells are used. In vitro assays can also include cell-free assays in which no intact cells are used.

[0121] As used herein, the term "about" refers to a number plus or minus 10% of that number. The term "about" refers to a range of minus 10% of the lowest value and plus 10% of the highest value of a range.

[0122] As used herein, the term "treatment" or "treating" is used in reference to a pharmaceutical or other intervention regimen to obtain a beneficial or desired result in a recipient. The beneficial or desired result includes, but is not limited to, a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to the eradication or amelioration of the condition or underlying disease being treated. A therapeutic benefit may also be achieved by the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or illness, delaying or eliminating the onset of symptoms of a disease or illness, delaying, halting, or reversing the progression of a disease or illness, or any combination thereof. For a prophylactic benefit, a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease may receive treatment, even if a diagnosis of the disease has not been made.

[0123] As used herein, the term "promoter" refers to a region or regions of a nucleic acid sequence that regulates transcription.

[0124] The term "regulatory element," as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and the like.

[0125] The term "vector" as used herein refers to a nucleic acid molecule (typically composed of DNA) that is capable of replicating in a host cell and / or to which another nucleic acid segment can be operably linked to effect replication of the attached segment. Plasmids, cosmids, or viruses are exemplary vectors. In some embodiments, the vector is an AAV plasmid.

[0126] As used herein, the phrases "substantially identical" or "substantially identical" in the context of two nucleic acid molecules, nucleotide sequences, or protein sequences, when compared and aligned for maximum correspondence, mean a sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ...9%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 1 Substantial identity refers to two or more sequences or subsequences having nucleotide or amino acid residue identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95, 96, 97, 98, or 99%. For sequence comparison, typically, one sequence acts as a reference sequence to which test sequence is compared. When using sequence comparison algorithm, test sequence and reference sequence are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Sequence comparison algorithm then calculates the percent sequence identity of test sequence to reference sequence based on designated program parameters.

[0127] The optimal alignment of sequences for aligning the comparison window is performed by tools such as Smith and Waterman local homology algorithm, Needleman and Wunsch homology alignment algorithm, Pearson and Lipman similarity search method, and optionally computerized implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA available as part of GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.). The "fraction of identity" of the aligned segments of the test sequence and the reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The percent sequence identity is expressed as the fraction of identity multiplied by 100. The comparison of one or more polynucleotide sequences can be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. In some instances, "percent identity" is determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0128] Percent (%) sequence identity to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, % amino acid sequence identity values ​​can be generated using the sequence comparison program ALIGN2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted with user documentation to the US Copyright Office, Washington, DC, 20559, and is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0129] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (or alternatively, the identity of a given amino acid sequence A to a given amino acid sequence B with a certain % amino acid sequence identity) is calculated as follows: multiply the fraction X / Y by 100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in sequences A and B of the program, and Y is the total number of amino acid residues in B. It is to be understood that the length of amino acid sequence A is not equal to the length of amino acid sequence B, and the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0130] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. EXAMPLES

[0131] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0132] Example 1 Loss of ITRs in Ampicillin and Antibiotic Resistant Cells The production of ITR plasmids is vulnerable to replication errors. ITRs are highly unstable in kanamycin-resistant adapted cell lines. AAV plasmids containing ITRs were amplified in kanamycin-resistant Endura cells and ampicillin-resistant SURE cells. The purified plasmids were linearized with SmaI, yielding two bands between 2000 and 3000 base pairs and three bands between 500 and 700 base pairs, as shown by lane 1 in Figure 1A. The loss of ITRs in the plasmids is represented by a 5000 bp band. The plasmids amplified in ampicillin-resistant cells (Figure 1B) showed a stronger loss of ITRs, as shown by the 5000 bp band, than the plasmids amplified in kanamycin-resistant cells (Figure 1C).

[0133] The polyG / C sequence shown in Figure 2A (arrow) was removed from the kanamycin resistance plasmid. In addition, the polyC run adjacent to the R-ITR was removed. The plasmid was digested with SmaI and run on a gel to identify the stability of the ITRS. As shown in Figure 2B, the ITR stability was improved by the removal of the polyG / C sequence (compare Figure 2B with Figure 1B).

[0134] Example 2 Prevention of reverse packaging using a stuffer sequence The efficiency of packaging rAAV cassettes is reduced by the production of empty capsids and reverse packaged backbone sequences, as shown in Figure 3. Stuffer sequences were designed to increase the size of the backbone to reduce reverse packaging. First, stuffer DNA sequences were randomly generated. Then, all open reading frames larger than 20 amino acids were mutated. Then, CpG islands were mutated until they were no longer detectable. Then, most restriction enzyme sites were removed. Then, repeat strings of more than 4 nucleobases were removed.

[0135] A stuffer sequence was inserted into the backbone at a site located 5' of the origin, as shown in Figure 4A. Plasmids were isolated and digested with SmaI to determine the effect on ITR stability. Larger ITR losses were observed, as shown in Figure 4B.

[0136] A second plasmid was designed as shown in Figure 5A. The stuffer is located 3' to the replication origin. The resistance gene was modified to the standard KanR. The M13 replication origin was removed. To determine ITR stability, the plasmid was isolated and digested with SmaI. Little or no ITR loss was observed, as shown in Figure 5B.

[0137] The polyG / C sequence was reintroduced into the plasmid shown in Figure 5B. The plasmid was amplified in kanamycin resistant cells, isolated, and digested with SmaI. As shown in Figure 6, ITR loss was observed, indicating that both the stuffer placement and polyG / C removal are necessary for enhanced ITR stability.

[0138] Example 3 Stability of modified transfer plasmid backbones The original transfer plasmid was amplified in SURE cells. The final transfer plasmid (Figure 5A) was amplified in Endura cells. 10 mg of DNA was produced and isolated. The plasmids were digested with either SmaI or AdhI. The original SmaI plasmid showed greater loss than the modified plasmid, as shown in Figure 7.

[0139] Example 4 Use of the three-plasmid system for expression of human retinoschisin Transfer plasmid This plasmid generates a single-stranded rAAV genome designed to express human retinoschisin (hRS1) in the retina. A de novo synthetic RS1 cDNA was generated containing four synonymous substitutions to facilitate restriction enzyme molecular cloning. Expression of human RS1 is driven specifically in rod and cone photoreceptors by the hGRK1 promoter linked to an SV40 splice donor / splice acceptor that promotes mRNA transport to the cytoplasm after removal of the SV40 intron. Translation is enhanced by incorporation of a consensus Kozak sequence immediately preceding the hRS1syn start codon, and transcript stability is enhanced by inclusion of a WPRE immediately following the hRS1syn open reading frame. The version of the WPRE used contains mutations designed to eliminate expression of the putative X protein ORF14,15. The expression cassette ends with a bovine growth hormone polyadenylation signal (bGH polyA).

[0140] The initial construction of the plasmid began by cloning into the previously used AAV vector plasmid containing the AAV2 ITR, hGRK1 promoter, SV40 SD / SA, and bGH polyA into human RS1syn as described above. WPRE was later cloned into the pJET-WPRE position to generate pTR-X002 (pTR-GRK1-RS1 syn-WPRE). The small size of the vector (2,31 lbp) was undesirable due to the possibility of aberrant packaging, therefore an inactive stuffer sequence was added 3' to the bGH polyA to generate a 4,549 bp vector cassette that approximates the optimal packaging size of the wild-type AAV genome. The 2,234 bp stuffer sequence was designed in silico using a random DNA generator (molbiotools.com) and curated to exclude all open reading frames (ORFs) larger than 20 amino acids in both the sense / antisense strands, depleted CpG islands, and excluded repeat sequences larger than 4 nucleotides. This sequence was synthesized de novo and cloned using SacESphI restriction enzyme cloning to place a stuffer sequence at the 3' end of the bGH polyA to generate pTR-X002-3p (Figure 8B). This cassette was then packaged into AAV.SPR to determine improved efficacy compared to the original unstuffed cassette in the RS1KO mouse model. To generate the final version of the plasmid (pTR-X002-3pSR), a kanamycin resistance backbone (from pUC57-KanR) was synthesized de novo to contain an additional stuffer sequence (distinct from the internal rAAV cassette stuffer) and cloned using two PacI restriction sites. This large backbone (5,808 bp ITR-ITR) was designed to prevent reverse packaging by exceeding the capacity of rAAV packaging. All plasmids were sequence verified by full-plasmid next-generation sequencing at the Massachusetts General Hospital Sequencing Core (Boston, MA).An annotated map of the pTR-X002-3pSR plasmid is shown in (Figure 8A) and a diagram illustrating the cloning strategy is shown in Figure 8B.

[0141] Vector stuffer molecule sequence A 2,234 bp stuffer sequence was designed in silico at Atsena Therapeutics using a random DNA generator (molbiotools.com) and curated to exclude all open reading frames (ORFs) larger than 20 amino acids in both the sense and antisense strands, deplete CpG islands, and exclude repetitive sequences larger than 4 nucleotides. This stuffer sequence and the backbone stuffer sequence are distinct and originate from different runs of the random DNA generator.

[0142] AAV-SPR Rep / Cap plasmid pC44.9(E531D)-R A precursor AAV2 rep-AAV.SPR plasmid, pCAAV.SPR, was constructed. pCAAV.SPR was constructed from pACG2 in which the AAV2 VP1 coding sequence was replaced with the AAV.SPR coding sequence. Both the rep and cap genes are under the control of the endogenous AAV promoter element. The AAV.SPR cap sequence encoding VP1 / VP2 / VP3 was generated by de novo synthesis (Genscript NJ).

[0143] The original pCAAV.SPR backbone contained an ampicillin selection marker (AmpR) and unnecessary legacy sequences associated with the construction of pACG2. The plasmid was modified using restriction enzyme cloning and ligation-independent cloning methods to remove unnecessary sequences and replace the origin of replication and AmpR with a kanamycin resistance (KanR) gene and origin of replication from pUC57-KanR (Genscript, NJ). This final pCAAV.SPR-R plasmid (Figure 8C) has been fully verified by next-generation sequencing at the Massachusetts General Hospital sequencing core and has been verified in a small-scale model to package correctly. An annotated map of the pCAAV.SPR-R plasmid is shown in Figure 8C.

[0144] Helper plasmid pALD-X80 The helper plasmid DNA provided cells with the E2a, E4, and VA RNA helper genes from adenovirus type 5 to support vector production without the need for wild-type virus coinfection. An annotated map of the pALD-X80 plasmid is shown in Figure 8D.

[0145] Example 5 Evaluation of hRS1-containing rAAV vectors with stuffer sequences This study was undertaken to evaluate optimized hRS1-containing AAV.SPR vectors with genome sizes that result in efficient packaging. The goal was to identify constructs that are at least as effective as rAAV.SPR-X001.

[0146] Due to the small packaging size of pTR-X001 (1723 bp) and the possibility of heterologous genome packaging, several new constructs were designed with cassette sizes approaching the natural carrying dose of AAV (~4.7 Kb ITR to ITR cassette). This was achieved by the addition of an inactive stuffer sequence (pTR-X001-5p and pTR-X001-3p, respectively) inserted within the vector cassette either 5' to (X001-5p) or 3' to (X001-3p) the hGRK1-hRS1-syn-bGH polyA. Stuffer DNA was synthesized de novo (Genscript, NJ). In addition, a version (pTR-X002-3p) was created that incorporated the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) located between hRS1syn and bGH polyA. The WPRE variants used have been previously incorporated into AAV vectors used in other ocular gene therapy clinical trials. rAAV.SPR vectors were produced by packaging these expression cassettes, and vector genomes ranged in size from 4534 to 4549 nucleotides. Multiple constructs evaluated are shown in Figures 9A-9C.

[0147] To test for restoration of retinal structure / function in RS1KO mice, we performed a 3-month preclinical study using two vector doses. The "stuffer-free" vector pTR-X001 (rAAV.SPR-X001), which rescued retinal structure / function in the previously described RS1KO mouse study, was included as a comparative control.

[0148] An overview of the cassette selection study design is shown in Table 1. RS1KO mice were subretinally injected in one eye with either vehicle (group 1), rAAV.SPR-X001 (groups 2 and 3), rAAV.SPR-X001-3p (groups 4 and 5), rAAV.SPR-X001-5p (groups 6 and 7), or rAAV.SPR-X002-3p (groups 8 and 9) vectors. Vectors were administered at 1.0 × 10 11 vg / mL; 8 vg / eye (groups 2, 4, 6, 8) or 5.0x1011 vg / mL; 5.0x10 8 vg / eye (groups 3, 5, 7, 9). The contralateral eye remained uninjected. Retinal structure and function were assessed by OCT and ERG, respectively, approximately 1 and 2 months after injection. Animals were euthanized approximately 3 months after injection. After euthanasia, retinas were cryosectioned and evaluated for RS1 expression via immunohistochemistry.

[0149] [Table 1]

[0150] OCT analysis revealed resolution of retinoschisis cavities in vector-treated eyes (Figure 10). There was a statically significant difference in retinoschisis scores between vehicle- and vector-treated eyes at both time points, except for eyes treated with the low dose of rAAV.SPR-X001-5p and evaluated 1 month after injection. 11 vg / mL (1.0 × 10 8 At a concentration of 5 × 10 vg / eye, rAAV.SPR-X002-3p resulted in significant improvement in rod- and cone-mediated function compared to eyes injected with vehicle alone (FIG. 11A). 11 vg / mL (5.0 × 10 8 At 100 mg / eye (100 mg / eye), all three stuffer constructs conferred significant improvement in retinal function compared to vehicle-injected controls (FIG. 11B). No significant differences in ERG amplitude between eyes injected with rAAV.SPR carrying the stuffer construct and eyes injected with rAAV.SPR carrying the non-stuffer construct were observed at this higher dose.

[0151] Immunohistochemistry revealed the presence of RS1 expression in the photoreceptor inner segments of retinas from RS1KO mouse eyes treated with rAAV.SPR-X002-3p, which was absent in contralateral untreated and vehicle-only treated eyes (Figures 12A-B).

[0152] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in carrying out the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

[0153] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13]

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Claims

**Claim 1**: A nucleic acid stuffer sequence comprising a sequence that is at least 80% identical to at least 100 consecutive bases of SEQ ID NO:

7. **Claim 2**: An adeno-associated virus (AAV) plasmid comprising the nucleic acid stuffer sequence according to Claim 1. **Claim 3**: The AAV plasmid according to Claim 2, wherein the nucleic acid stuffer sequence has a length of about 100 to about 5000 nucleic acid bases. **Claim 4**: The AAV plasmid according to Claim 2, further comprising a polyA sequence, wherein the nucleic acid stuffer sequence is located downstream of the polyA sequence. **Claim 5**: The AAV plasmid according to Claim 2, wherein the AAV plasmid comprises an expression cassette containing a heterologous sequence located between two inverted terminal repeat (ITR) sequences. **Claim 6**: The AAV plasmid according to Claim 5, wherein the expression cassette has a length of about 3000 to about 6000 nucleic acid bases. **Claim 7**: The AAV plasmid according to Claim 5, wherein the heterologous sequence encodes a therapeutic peptide, and the therapeutic peptide is RS1, GUCY2D, MYO7A, CNGB3, ADAMTS10, ABCA4, or frataxin. **Claim 8**: A nucleic acid stuffer sequence comprising a sequence that is at least 80% identical to at least 100 consecutive bases of SEQ ID NO:

8. **Claim 9**: An AAV plasmid comprising the nucleic acid stuffer sequence according to Claim 8, wherein the AAV plasmid comprises an expression cassette containing a heterologous sequence located between two inverted terminal repeat sequences, and the nucleic acid stuffer sequence is located outside the expression cassette. **Claim 10**: The AAV plasmid according to Claim 9, wherein the nucleic acid stuffer sequence is located between the origin of replication and one of the inverted terminal repeat sequences such that one of the inverted terminal repeat sequences is about 1000 to about 4000 nucleic acid bases away from the origin of replication. **Claim 11**: A nucleic acid stuffer sequence comprising a sequence that is at least 80% identical to at least 100 consecutive bases of SEQ ID NO:

11. **Claim 12**: An AAV plasmid comprising the nucleic acid stuffer sequence according to Claim 11, wherein the AAV plasmid comprises an expression cassette containing a heterologous sequence located between two inverted terminal repeat sequences, and the nucleic acid stuffer sequence is located outside the expression cassette. **Claim 13**: The AAV plasmid according to claim 12, wherein the nucleic acid stuffer sequence is positioned such that one of the inverted terminal repeat sequences is about 1000 to about 4000 nucleobases away from the origin of replication. **Claim 14**: The AAV plasmid according to any one of claims 5-7, 9-10, or 12-13, wherein the presence of the nucleic acid stuffer sequence reduces the mutation of one or both of the inverted terminal repeat sequences as compared to an AAV plasmid that does not contain the nucleic acid stuffer sequence. **Claim 15**: The AAV plasmid according to claim 14, further comprising a kanamycin resistance gene. **Claim 16**: The AAV plasmid according to claim 14, and (a) a packaging plasmid comprising a viral replication (rep) gene, a viral capsid (cap) gene, or both, (b) a helper plasmid, or (c) both (a) and (b) A composition comprising. **Claim 17**: A host cell comprising the composition according to claim 16. **Claim 18**: An AAV particle comprising a nucleic acid and a capsid, wherein the AAV particle is produced by the host cell according to claim 17. **Claim 19**: A method for transducing a cell, the method comprising administering the AAV particle according to claim 18 to the cell. **Claim 20**: A pharmaceutical composition for treating an eye disease or disorder in a mammal, the pharmaceutical composition comprising the AAV particle according to claim 18.

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