Additives for viral vector production

Abstract

Among other things, the present disclosure provides methods, systems, and compositions for production of viral vectors, e.g., recombinant viral vectors, e.g., recombinant adeno-associated viruses (rAAVs). In some embodiments, a method comprises addition of one or more additives to a medium comprising host cells. In some embodiments, an addition of an additive to a medium can provide increased titer of produced recombinant viral vector particles, e.g., rAAV particles. In some embodiments, an additive comprises momelotinib or a salt or derivative thereof.

Description

Attorney Docket No.: 2011256-2625ADDITIVES FOR VIRAL VECTOR PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63 / 693,997, filed September 12, 2024, the entirety of which is incorporated herein by reference.BACKGROUND

[0002] Recombinant viral vectors, such as recombinant adeno-associated viruses (rAAVs), are an important delivery vector for gene therapy for treatment of a wide variety of diseases. Development of recombinant viral vectors, e.g., rAAVs, and the therapeutic payloads contained therein is rapidly proceeding. However, there remains a need for improved methods, systems, and compositions for manufacturing recombinant viral vectors, e.g., rAAVs, to effectively produce high quantities of high quality recombinant viral vectors in order to support implementation of related gene therapies in clinics. In particular, such need includes methods, systems, and compositions for increasing the titer of recombinant viral vector particles produced.SUMMARY

[0003] Among other things, the present disclosure provides methods for producing a plurality of recombinant adeno-associated virus (rAAV) particles. In some embodiments, the present disclosure provides a method for producing a plurality of rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors; (b) adding an additive to the medium; and (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles. In some embodiments, the present disclosure provides a method for producing a plurality of rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors encoding (i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one Rep polypeptide, (iii) at least one Cap polypeptide, and (iv) at least one helper polypeptide; (b) adding an additive to the medium; and (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles. In some embodiments, a method for producing a plurality of rAAV particles further comprises collecting the produced plurality of rAAV particles from the culture. In some embodiments, the present disclosure provides a method for producing a plurality of rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles, and (d) collecting the produced plurality of rAAV particles from the culture. In some embodiments, the present disclosure provides aPage 1 of 10712963423v 1Attorney Docket No.: 2011256-2625 method for producing a plurality of rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors encoding (i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one Rep polypeptide, (iii) at least one Cap polypeptide, and (iv) at least one helper polypeptide; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles; and (d) collecting the produced plurality of rAAV particles from the culture.

[0004] In some embodiments, a plurality of rAAV particles are produced at an increased titer as compared to a plurality of rAAV particles produced without an additive added to a medium. In some embodiments, an increased titer is measured at about 1 to about 10 days after transfection. In some embodiments, an increased titer is measured at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after transfection. In some embodiments, an increased titer comprises or is an about 1-fold to about 3-fold, about 1-fold to about 2-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 2-fold, or about 2-fold to about 2.5- fold increased titer. In some embodiments, an increased titer comprises or is an about 1-fold to about 3- fold increased titer. In some embodiments, an increased titer comprises or is an about 1-fold or more, 1.25- fold or more, 1.5-fold or more, 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, or 3 -fold or more increased titer. In some embodiments, an increased titer comprises or is an about at least 1- fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, or at least 3-fold increased titer.

[0005] In some embodiments, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising a plurality of rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles; (d) collecting the produced plurality of rAAV particles from the culture; and (e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles. In some embodiments, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising a plurality of rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors encoding (i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one Rep polypeptide, (iii) at least one Cap polypeptide, and (iv) at least one helper polypeptide; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles; (d) collecting the produced plurality of rAAV particles from the culture; and (e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles. In some embodiments, the present disclosure further comprisesPage 2 of 10712963423v 1Attorney Docket No.: 2011256-2625 purifying the produced plurality of rAAV particles prior to adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers. In some embodiments, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising a plurality of rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles; (d) collecting the produced plurality of rAAV particles from the culture; (e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles; and (f) purifying the produced plurality of rAAV particles prior to adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers. In some embodiments, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising a plurality of rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors encoding (i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one Rep polypeptide, (iii) at least one Cap polypeptide, and (iv) at least one helper polypeptide; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles; (d) collecting the produced plurality of rAAV particles from the culture; (e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles; and (f) purifying the produced plurality of rAAV particles prior to adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers.

[0006] In some embodiments, an additive comprises or is a Janus kinase (JAK) inhibitor, a phosphoinositide-dependent kinase- 1 (PDKl) / protein kinase B (AKT) signaling inhibitor, a TANK-binding kinase 1 (TBK1) inhibitor, an interferon inhibitor, an IKB kinase (IKK) inhibitor, a NF-KB inhibitor, a MNK1 inhibitor, a proteasome inhibitor, a protein neddylation inhibitor, a histone deacetylase (HDAC) inhibitor, or a protein kinase R (PKR) inhibitor or a combination thereof. In some embodiments, an additive comprises or is a JAK inhibitor. In some embodiments, an additive comprises or is a PDK1 / AKT signaling inhibitor. In some embodiments, an additive comprises or is a TBK1 inhibitor. In some embodiments, an additive comprises or is an interferon inhibitor. In some embodiments, an additive comprises or is an IKK inhibitor. In some embodiments, an additive comprises or is a NF-KB inhibitor. In some embodiments, an additive comprises or is a MNK1 inhibitor. In some embodiments, an additive comprises or is a proteasome inhibitor. In some embodiments, an additive comprises or is a protein neddylation inhibitor. In some embodiments, an additive comprises or is a HDAC inhibitor. In some embodiments, an additive comprises or is a PKR inhibitor. In some embodiments, an additive is a combination of any of the foregoing.Page 3 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0007] In some embodiments, an additive comprises or is momelotinib, ruxolitinib, BX-795, interferon-a-IFNa-R interaction inhibitor, BMS-345541, BAY 11-7082, CGP 57380, epoxomicin, IMD 0354, imidazolo-oxindole, lactacystin, MLN-4924, NF-KB activation inhibitor, parthenolide, spermine, tosylate salt, TPCA-1, or trichostatin A, or a salt or derivative thereof. In some embodiments, an additive comprises or is momelotinib or a salt or derivative thereof. In some embodiments, an additive comprises or is ruxolitinib or a salt or derivative thereof. In some embodiments, an additive comprises or is BX-795 or a salt or derivative thereof. In some embodiments, an additive comprises or is interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof. In some embodiments, an additive comprises or is BMS- 345541 or a salt or derivative thereof. In some embodiments, an additive comprises or is BAY 11-7082 or a salt or derivative thereof. In some embodiments, an additive comprises or is CGP 57380 or a salt or derivative thereof. In some embodiments, an additive comprises or is epoxomicin or a salt or derivative thereof. In some embodiments, an additive comprises or is IMD 0354 or a salt or derivative thereof. In some embodiments, an additive comprises or is imidazolo-oxindole or a salt or derivative thereof. In some embodiments, an additive comprises or is lactacystin or a salt or derivative thereof. In some embodiments, an additive comprises or is MLN-4924 or a salt or derivative thereof. In some embodiments, an additive comprises or is NF-KB activation inhibitor or a salt or derivative thereof. In some embodiments, an additive comprises or is parthenolide or a salt or derivative thereof. In some embodiments, an additive comprises or is spermine or a salt or derivative thereof. In some embodiments, an additive comprises or is tosylate salt or a derivative thereof. In some embodiments, an additive comprises or is TPCA-1 or a salt or derivative thereof. In some embodiments, an additive comprises or is trichostatin A or a salt or derivative thereof.

[0008] In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, about 1 pM to about 2 pM, about 1.5 pM to about 25 pM, about 1.5 pM to about 20 pM, about 1.5 pM to about 15 pM, about 1.5 pM to about 10 pM, about 1.5 pM to about 5 pM, about 1.5 pM to about 2.5 pM, or about 1.5 pM to about 2 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 0.5 to about 20 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7Page 4 of 10712963423v 1Attorney Docket No.: 2011256-2625 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about1.5 pM.

[0009] In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, or about 1 pM to about 2 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 0.5 to about 20 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM.

[0010] In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM to about 10, about 0.1 pM to about 7.5, about 0.1 pM to about 5, about 0.1 pM to about 4, about 0.1 pM to about 3, about 0.1 pM to about 2, about 0.1 pM to about 1, about 0.25 pM to about 10, about 0.25 pM to about 7.5, about 0.25 pM to about 5, about 0.25 pM to about 4, about 0.25 pM to about 3, about 0.25 pM to about 2, about 0.25 pM to about 1, about 0.5 pM to about 10, about 0.5 pM to about 7.5, about 0.5 pM to about 5, about 0.5 pM to about 4, about 0.5 pM to about 3, about 0.5 pM to about 2, about 0.5 pM to about 1, about 0.75 pM to about 10, about 0.75 pM to about 7.5, about 0.75 pM to about 5, about 0.75 pM to about 4, about 0.75 pM to about 3, about 0.75 pM to about 2, about 0.75 pM to about 1 1 pM to about 10, about 1 pM to about 7.5 1 pM to about 5, about 1 pM to about 4, about 1 pM to about 3, or about 1 pM to about 2 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.25 pM, about 1.5 pM, about 1.75 pM, about 2 pM, about 2.5 pM, about 3 pM, about3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or about 10 pM.

[0011] In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereofPage 5 of 10712963423v 1Attorney Docket No.: 2011256-2625 is added to a medium to a concentration of about 0.1 pM to about 25 M, about 0.1 |iM to about 20 pM, about 0.1 pM to about 15 pM, about 0.1 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 2.5 pM, about 0.1 pM to about 1 pM, about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM. about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, or about 1 pM to about 2.5 pM. In some embodiments, interferon-a- IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM, about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM. about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM.

[0012] In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM to about 25 pM, about 0.1 pM to about 20 pM, about 0.1 pM to about 15 pM, about 0.1 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 2.5 pM, about 0.1 pM to about 1 pM, about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, or about 1 pM to about 2.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM, about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM.

[0013] In some embodiments, an additive is added to a medium prior to transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium about 1 minute to about 60Page 6 of 10712963423v 1Attorney Docket No.: 2011256-2625 minutes, about 15 minutes to about 60 minutes, about 30 minutes to about 60 minutes, about 45 minutes to about 60 minutes, about 1 minute to about 45 minutes, about 15 minutes to about 45 minutes, about 30 minutes to about 45 minutes, about 1 minute to about 30 minutes, about 15 minutes to about 30 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, or 1 minute to about 5 minutes or about 1 hour to about 48 hours, about 6 hours to about 48 hours, about 12 hours to about 48 hours, about 18 hours to about 48 hours, about 24 hours to about 48 hours, about 30 hours to about 48 hours, about 36 hours to about 48 hours, about 42 hours to about 48 hours, about 1 hour to about 42 hours, about 6 hours to about 42 hours, about 12 hours to about 42 hours, about 18 hours to about 42 hours, about 24 hours to about 42 hours, about 30 hours to about 42 hours, about 36 hours to about 42 hours, about 1 hour to about 36 hours, about 6 hours to about 36 hours, about 12 hours to about 36 hours, about 18 hours to about 36 hours, about 24 hours to about 36 hours, about 30 hours to about 36 hours, about 1 hour to about 30 hours, about 6 hours to about 30 hours, about 12 hours to about 30 hours, about 18 hours to about 30 hours, about 24 hours to about 30 hours, about 1 hour to about 24 hours, about 6 hours to about 24 hours, about 12 hours to about 24 hours, about 18 hours to about 24 hours, about 1 hour to about 18 hours, about 6 hours to about 18 hours, about 12 hours to about 18 hours, about 1 hour to about 12 hours, about 6 hours to about 12 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, or about 1 hour to about 2 hours prior to transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium about 1 to about 48 hours prior to transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes or about 1 hour-, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or 48 hours prior to transfection prior to transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes or at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours prior to transfection of host cells with one or more vectors.

[0014] In some embodiments, an additive is added to a medium substantially simultaneously toPage 7 of 10712963423v 1Attorney Docket No.: 2011256-2625 transfection of host cells with one or more vectors.

[0015] In some embodiments, an additive is added to a medium after transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium about 1 minute to about 60 minutes, about 15 minutes to about 60 minutes, about 30 minutes to about 60 minutes, about 45 minutes to about 60 minutes, about 1 minute to about 45 minutes, about 15 minutes to about 45 minutes, about 30 minutes to about 45 minutes, about 1 minute to about 30 minutes, about 15 minutes to about 30 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, or about 1 minute to about 5 minutes or about 1 hour to about 24 hours, about 2 hours to about 24 hours, about 4 hours to about 24 hours, about 8 hours to about 24 hours, about 12 hours to about 24 hours, about 16 hours to about 24 hours, about 20 hours to about 24 hours, about 1 hour to about 20 hours, about 2 hours to about 20 hours, about 4 hours to about 20 hours, about 8 hours to about 20 hours, about 12 hours to about 20 hours, about 16 hours to about 20 hours, about 1 hour to about 16 hours, about 2 hours to about 16 hours, about 4 hours to about 16 hours, about 8 hours to about 16 hours, about 12 hours to about 16 hours, about 1 hour to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 8 hours to about 12 hours, about 1 hour to about 8 hours, about 2 hours to about 8 hours, about 4 hours to about 8 hours, about 1 hour to about4 hours, about 2 hours to about 4 hours or about 1 hour to about 2 hours after transfection with one or more vectors. In some embodiments, an additive is added to a medium about 1 to about 60 minutes after transfection with one or more vectors. In some embodiments, an additive is added to a medium about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes or about 1 hour, about 2 hours, about 3 hour's, about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours after transfection with one or more vectors. In some embodiments, an additive is added to a medium at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes or at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours after transfection with one or more vectors.

[0016] In some embodiments, a method for producing a plurality of rAAV particles further comprises adding one or more additional additives. In some embodiments, one or more additional additives comprises or is a microtubule destabilizer. In some embodiments, a microtubule destabilizer comprises or is a G2 / MPage 8 of 10712963423v 1Attorney Docket No.: 2011256-2625 inhibitor. In some embodiments, one or more additional additives comprises or is a microtubule destabilizer, optionally wherein the microtubule destabilizer comprises or is a G2 / M inhibitor. In some embodiments, a microtubule destabilizer comprises or is colcemid, colchicine, vinblastine, or a salt or derivative thereof. In some embodiments, a microtubule destabilizer comprises or is colcemid or a salt or derivative thereof. In some embodiments, a microtubule destabilizer comprises or is colchicine or a salt or derivative thereof. In some embodiments, a microtubule destabilizer comprises or is vinblastine or a salt or derivative thereof. In some embodiments, one or more additional additives are added to a medium prior to transfection of host cells with one or more vectors. In some embodiments, one or more additional additives are added to a medium substantially simultaneously to transfection of host cells with one or more vectors. In some embodiments, one or more additional additives are added to a medium after transfection of host cells with one or more vectors. In some embodiments, one or more additional additives are added to a medium prior to addition of an additive. In some embodiments, one or more additional additives are added to a medium substantially simultaneously to addition of an additive. In some embodiments, one or more additional additives are added to a medium after addition of an additive.

[0017] In some embodiments, host cells comprise or are adherent cells. In some embodiments, host cells comprise or are suspension cells. In some embodiments, host cells comprise or are mammalian cells. In some embodiments, host cells comprise or are HEK293 cells, CHO cells, HeLa cells, or a variant thereof. In some embodiments, host cells comprise or are HEK293 cells or a variant thereof. In some embodiments, host cells comprise or are CHO cells or a variant thereof. In some embodiments, host cells comprise or are HeLa cells or a variant thereof.

[0018] In some embodiments, one or more vectors comprise (i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload, (ii) a second vector encoding at least one Rep polypeptide and at least one Cap polypeptide, and (iii) a third vector encoding at least one helper polypeptide. In some embodiments, one or more vectors comprise (i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload and (ii) a second vector encoding at least one Rep polypeptide, at least one Cap polypeptide, and at least one helper polypeptide. In some embodiments, one or more vectors comprise (i) a first vector encoding at least one Cap polypeptide and at least one payload flanked by an ITR on either side of the at least one payload and (ii) a second vector encoding at least one Rep polypeptide and at least one helper polypeptide. In some embodiments, a helper polypeptide comprises one, two, three, or four of El, E2A, E4orf6, or VA RNA polypeptides.

[0019] In some embodiments, one or more vectors are transfected into host cells in the presence of a transfection reagent. In some embodiments, a transfection reagent comprises or is a cationic polymer. In some embodiments, a cationic polymer comprises or is polycthylcniminc. In some embodiments, one or more vectors are transfected into host cells in the presence of a cationic polymer. In some embodiments,Page 9 of 10712963423v 1Attorney Docket No.: 2011256-2625 one or more vectors are transfected into host cells in the presence of polyethylenimine (PEI). In some embodiments, one or more vectors are transfected into host cells in the presence of calcium phosphate. In some embodiments, one or more vectors are transfected into host cells in the presence of cationic liposomes. In some embodiments, one or more vectors are transfected into host cells in the presence of FectoVIR (e.g., FectoVIR-AAV) transfection reagent. In some embodiments, one or more vectors are transfected into host cells in the presence of Lipofectamine (e.g., Lipofectamine, Lipofectamine 2000, Lipofectamine 3000) transfection reagent. In some embodiments, one or more vectors are transfected into host cells in the presence of TransIT-VirusGEN transfection reagent.

[0020] In some embodiments, the present disclosure provides a reaction mixture. In some embodiments, the present disclosure provides a reaction mixture comprising (a) one or more vectors and (b) an additive. In some embodiments, the present disclosure provides a reaction mixture comprising (a) one or more vectors encoding (i) at least one payload flanked by an ITR on either side of the at least one payload, (ii) at least one Rep polypeptide, (iii) at least one Cap polypeptide, and (iv) at least one helper polypeptide and (b) an additive. In some embodiments, one or more vectors comprise (i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload, (ii) a second vector encoding at least one Rep polypeptide and at least one Cap polypeptide, and (iii) a third vector encoding at least one helper polypeptide. In some embodiments, one or more vectors comprise (i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload and (ii) a second vector encoding at least one Rep polypeptide, at least one Cap polypeptide, and at least one helper polypeptide. In some embodiments, one or more vectors comprise (i) a first vector encoding at least one Cap polypeptide and at least one payload flanked by an ITR on either side of the at least one payload and (ii) a second vector encoding at least one Rep polypeptide and at least one helper polypeptide.

[0021] In some embodiments, an additive in a reaction mixture comprises or is a Janus kinase (JAK) inhibitor, a phosphoinositide-dependent kinase-1 (PDK1 ) / protein kinase B (AKT) signaling inhibitor, a TANK-binding kinase 1 (TBK1 ) inhibitor, an interferon inhibitor, an IKB kinase (IKK) inhibitor, a NF-KB inhibitor, a MNK1 inhibitor, a proteasome inhibitor, a protein neddylation inhibitor, a histone deacetylase (HD AC) inhibitor, or a protein kinase R (PKR) inhibitor or a combination thereof. In some embodiments, an additive in a reaction mixture comprises or is momelotinib, ruxolitinib, BX-795, interferon- a-IFNa-R interaction inhibitor, BMS-345541, BAY 11-7082, CGP 57380, epoxomicin, IMD 0354, imidazolo- oxindole, lactacystin, MLN-4924, NF-KB activation inhibitor, parthenolide, spermine, tosylate salt, TPCA- 1, or trichostatin A, or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is ruxolitinib or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is BX-795 or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof.Page 10 of 10712963423v 1Attorney Docket No.: 2011256-2625In some embodiments, an additive in a reaction mixture comprises or is BMS-345541 or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is BAY 11-7082 or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is CGP 57380 or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is epoxomicin or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is IMD 0354 or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is imidazolo-oxindole or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is lactacystin or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is MLN-4924 or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is NF-KB activation inhibitor or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is parthenolide or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is spermine or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is tosylate salt or a derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is TPCA-1 or a salt or derivative thereof. In some embodiments, an additive in a reaction mixture comprises or is trichostatin A or a salt or derivative thereof.

[0022] In some embodiments, the present disclosure provides a culture comprising a plurality of host cells and a reaction mixture provided herein. In some embodiments, host cells comprise or are adherent cells. In some embodiments, host cells comprise or are suspension cells. In some embodiments, host cells comprise or are mammalian cells. In some embodiments, host cells comprise or are HEK293 cells, CHO cells, HeLa cells, or a variant thereof. In some embodiments, host cells comprise or are HEK293 cells or a valiant thereof. In some embodiments, host cells comprise or are CHO cells or a valiant thereof. In some embodiments, host cells comprise or are HeLa cells or a variant thereof.

[0023] In some embodiments, the present disclosure provides a bioreactor comprising a culture provided herein. In some embodiments, a bioreactor comprises or is a batch culture bioreactor, continuous culture bioreactor, fed batch culture bioreactor, or perfusion culture bioreactor. In some embodiments, a bioreactor comprises or is a batch culture bioreactor. In some embodiments, a bioreactor comprises or is a continuous culture bioreactor. In some embodiments, a bioreactor comprises or is a fed batch culture bioreactor. In some embodiments, a bioreactor comprises or is a perfusion culture bioreactor.

[0024] In some embodiments, the present disclosure provides a method for producing a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least on Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; and (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles. In some embodiments, the present disclosure provides a method for producing a plurality ofPage 11 of 10712963423v 1Attorney Docket No.: 2011256-2625 rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least on Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; and (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles; wherein the one or more host cells are each independently a producer cell that comprises (i) a nucleic acid sequence encoding at least one payload flanked by an ITR on either side of the at least one payload; (ii) a nucleic acid sequence encoding at least one Rep polypeptide; and (iii) a nucleic acid sequence encoding at least one Cap polypeptide. In some embodiments, a method for producing a plurality of rAAV particles further comprises collecting the produced plurality of rAAV particles from the culture. In some embodiments, the present disclosure provides a method for producing a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least on Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles; and (d) collecting the produced plurality of rAAV particles from the culture. In some embodiments, the present disclosure provides a method for producing a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least on Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles; and (d) collecting the produced plurality of rAAV particles from the culture; wherein the one or more host cells are each independently a producer cell that comprises (i) a nucleic acid sequence encoding at least one payload flanked by an ITR on either side of the at least one payload; (ii) a nucleic acid sequence encoding at least one Rep polypeptide; and (iii) a nucleic acid sequence encoding at least one Cap polypeptide.

[0025] In some embodiments, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least one Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles; (d) collecting the produced plurality of rAAV particles from the culture; and (e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles. In some embodiments, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least one Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles; (d) collecting the produced plurality of rAAV particles from the culture; and (e) adding one or more pharmaceutically acceptablePage 12 of 10712963423v 1Attorney Docket No.: 2011256-2625 diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles; wherein the one or more host cells are each independently a producer cell that comprises (i) a nucleic acid sequence encoding at least one payload flanked by an ITR on either side of the at least one payload; (ii) a nucleic acid sequence encoding at least one Rep polypeptide; and (iii) a nucleic acid sequence encoding at least one Cap polypeptide. In some embodiments, the present disclosure further comprises purifying the produced plurality of rAAV particles prior to adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers. In some embodiments, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least one Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles; (d) collecting the produced plurality of rAAV particles from the culture; (e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles; and (f) purifying the produced plurality of rAAV particles prior to adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers. In some embodiments, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least one Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles; (d) collecting the produced plurality of rAAV particles from the culture; (e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles; and (I) purifying the produced plurality of rAAV particles prior to adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers; wherein the one or more host cells are each independently a producer cell that comprises (i) a nucleic acid sequence encoding at least one payload flanked by an ITR on either side of the at least one pay load; (ii) a nucleic acid sequence encoding at least one Rep polypeptide; and (iii) a nucleic acid sequence encoding at least one Cap polypeptide.

[0026] In some embodiments, inducing expression of at least one Rep polypeptide and at least one Cap polypeptide comprises expressing at least one helper polypeptide in the one or more host cells. In some embodiments, inducing expression of at least one Rep polypeptide and at least one Cap polypeptide comprises contacting the one or more host cells with at least one helper virus.Page 13 of 10712963423v 1Attorney Docket No.: 2011256-2625BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1. Various compounds can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml in 5 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in a 24-deep well plate. Cells were transfected on day 2 with a pay load vector, a Rep / Cap vector, and a helper vector. Various compounds (BX-795, parthenolide, ruxolitinib, TPCA-1, momelotinib (CYT387), trichostatin A (TSA)) were added to the cultures at various concentrations (for BX-795, parthenolide, ruxolitinib, TPCA-1, and momelotinib, 0.5, 1, 4, and 10 pM; for TSA, 0.05, 0.1, 0.4, and 1 pM) at either 30 min post-transfection or 24 hrs post-transfection. Control cultures that did not receive any compound and control cultures that received only DMSO at various concentrations (0.05, 0.1, 0.4, and 1 pL / mL) were also examined. At 3 days post-addition of the various compounds, cells were harvested and lysed and resulting lysates were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of titer fold-change as compared to average titer from control cultures that did not receive any compound, with left graph showing results from addition at 30 min post-transfection and right graph showing results from addition at 24 hrs post-transfection. For each graph, for each compound listed along the X-axis, columns above correspond, from left to right, to data for 0.5, 1, 4, and 10 pM for BX-795, parthenolide, ruxolitinib, TPCA-1, and momelotinib; 0.05, 0.1, 0.4, and 1 pM for TSA; and 0.05, 0.1, 0.4, and 1 pL / mL for DMSO.

[0028] Figure 2. BX-795 and momelotinib can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml in 5 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in a 24-deep well plate. Cells were transfected on day 2 at a cell density of approximately 4 x 106viable cells / ml with a pay load vector, a Rep / Cap vector, and a helper vector. BX-795 or momelotinib (CYT387) was added to the cultures at various concentrations (for BX-795: 0.1, 0.2, 0.8, 1.7, and 3.3 pM; for momelotinib: 0.125, 0.25, 1, 2.5, and 5 pM) at 30 min post-transfection. Control cultures that did not receive any compound were also examined. At 4 days post-addition of the various compounds, cells were harvested and lysed and resulting lysates were supplemented with MgCb, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graph displays exemplary data of titer fold-change as compared to average titer from control cultures that did not receive any compound. For BX-795, columns on the graph correspond, from left to right, to data for 0.1, 0.2, 0.8, 1.7, and 3.3 pM. For momelotinib (CYT387), columns on the graph correspond, from left to right, to data for 0.125, 0.25, 1, 2.5, and 5 pM.

[0029] Figure 3. Various compounds can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml in 5 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in a 24-deep well plate.Page 14 of 10712963423v 1Attorney Docket No.: 2011256-2625Cells were transfected on day 2 with a payload vector, a Rep / Cap vector, and a helper vector. At 30 min post-transfection, various compounds were added to the cultures at various concentrations as shown on the graphs. Control cultures that did not receive any compound were also examined. At 3- or 4-days postaddition of the various compounds, cells were harvested and lysed and resulting lysates were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of titer for compound listed at top as normalized to control cultures that did not receive any compound. The horizontal line at 1.0 (or 1.00) on each graph represents the control cultures.

[0030] Figure 4. Various compounds can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml in 15 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a payload vector, a Rep / Cap vector, and a helper vector. At 30 min post-transfection, various compounds were added to the cultures at various concentrations as shown on the graphs. Control cultures that did not receive any compound were also examined. At 3 days post-addition of the various compounds, cells were harvested and lysed and resulting lysates were supplemented with MgClj, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of titer for compound listed at top. A horizontal line, labeled “Control”, on each graph represents the observed titer from control cultures.

[0031] Figure 5. Momelotinib can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml for AAV2 production or 0.75 x 106cells / ml for AAV9 production in 15 ml Freestyle Fl 7 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 P188 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a payload vector, a Rep / Cap vector, and a helper vector. At 30 min post-transfection, momelotinib was added to the cultures at various concentrations as shown on the graphs. A control culture without momelotinib added was also examined. At 4 days post-transfection, cells were harvested and lysed and resulting lysates from cells for production of AAV2 were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of genome titer (Y-axis) across tested concentrations of momelotinib (X-axis). A horizontal line, labeled “No Momelotinib Addition”, on each graph represents the observed titer from the control culture. The left graph shows data for AAV2 serotype rAAV particles, and the right graph shows data for AAV9 serotype rAAV particles.

[0032] Figure 6. Ruxolitinib can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml for AAV2 production or 0.75 x 106cells / ml for AAV9 production in 15 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 P188 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a payload vector,Page 15 of 10712963423v 1Attorney Docket No.: 2011256-2625 a Rep / Cap vector, and a helper vector. At 30 min post-transfection, ruxolitinib was added to the cultures at various concentrations as shown on the graphs. A control culture without ruxolitinib added was also examined. At 4 days post-transfection, cells were harvested and lysed and resulting lysates from cells for production of AAV2 were supplemented with MgCb, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of genome titer (Y-axis) across tested concentrations of ruxolitinib (X-axis). A horizontal line, labeled “No Ruxolitinib Addition”, on each graph represents the observed titer from the control culture. The left graph shows data for AAV2 serotype rAAV particles, and the right graph shows data for AAV9 serotype rAAV particles.

[0033] Figure 7. BX-795 can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml for AAV2 production or 0.75 x 106cells / ml for AAV9 production in 15 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a payload vector, a Rep / Cap vector, and a helper vector. At 30 min post-transfection, BX-795 was added to the cultures at various concentrations as shown on the graphs. A control culture without BX-795 added was also examined. At 4 days post-transfection, cells were harvested and lysed and resulting lysates from cells for production of AAV2 were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of genome titer (Y-axis) across tested concentrations of BX-795 (X-axis). A horizontal line, labeled “No BX-795 Addition”, on each graph represents the observed titer from the control culture. The left graph shows data for AAV2 serotype rAAV particles, and the right graph shows data for AAV9 serotype rAAV particles.

[0034] Figure 8. Momelotinib can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 0.75 x 106cells / ml in 15 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a pay load vector (encoding either pay load #1 or pay load #2), a Rep / Cap vector, and a helper vector. At 30 min post-transfection, momelotinib was added to the cultures at various concentrations as shown on the graphs. A control culture without momelotinib added was also examined. At 4 days post-transfection, cells were harvested and lysed. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of genome titer (Y-axis) across tested concentrations of momelotinib (X-axis). Horizontal dashed line represents genome titer from control culture without momelotinib. Left graph represents data from transfection with payload vector encoding payload #1; right graph represents data from transfection with payload vector encoding payload #2. * indicates that the genome titer was greater than the detection limit of 1 x 1012vg / ml.

[0035] Figure 9. Ruxolitinib can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 0.75 x 106cells / ml in 15 ml Freestyle F17 mediaPage 16 of 10712963423v 1Attorney Docket No.: 2011256-2625(Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a payload vector (encoding either payload #1 or payload #2), a Rep / Cap vector, and a helper vector. At 30 min post-transfection, ruxolitinib was added to the cultures at various concentrations as shown on the graphs. A control culture without ruxolitinib added was also examined. At 4 days post-transfection, cells were harvested and lysed. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of genome titer (Y-axis) across tested concentrations of ruxolitinib (X-axis). Horizontal dashed line represents genome titer from control culture without ruxolitinib. Left graph represents data from transfection with payload vector encoding payload #1 ; right graph represents data from transfection with payload vector encoding payload #2. * indicates that the genome titer was greater than the detection limit of 1 x 1012vg / ml.

[0036] Figure 10. BX-795 can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 0.75 x 106cells / ml in 15 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a payload vector (encoding either payload #1 or payload #2), a Rep / Cap vector, and a helper vector. At 30 min post-transfection, BX-795 was added to the cultures at various concentrations as shown on the graphs. A control culture without BX-795 added was also examined. At 4 days post-transfection, cells were harvested and lysed. rAAV genome titer was determined using ddPCR of the resultant lysates. Graphs display exemplary data of genome titer (Y-axis) across tested concentrations of BX-795 (X-axis). Horizontal dashed line represents genome titer from control culture without BX-795. Left graph represents data from transfection with payload vector encoding payload #1 ; right graph represents data from transfection with payload vector encoding payload #2.

[0037] Figure 11. Momelotinib can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 0.75 x 106cells / ml in 15 ml Freestyle F17 media (Thermo Fisher ) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in a production-scale bioreactor. Cells were transfected on day 2 at a cell density of approximately 3.5 x 106viable cells / ml with a payload vector, a Rep / Cap vector, and a helper vector. At 30 min post-transfection, momelotinib was added to the cultures at various concentrations as shown on the graph. A control culture without momelotinib added was also examined. At 4 days post-transfection, cells were harvested and lysed and resulting lysates were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graph displays exemplary data of genome titer (Y-axis) across tested concentrations of momelotinib (X-axis).

[0038] Figure 12. Momelotinib can modulate titers of produced rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 0.75 x 106cells / ml in 15 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in a production-scale bioreactor. CellsPage 17 of 10712963423v 1Attorney Docket No.: 2011256-2625 were transfected on day 2 at a cell density of approximately 3.5 x 106viable cells / ml with a payload vector, a Rep / Cap vector, and a helper vector. Momelotinib (0.91 M) was added to the cultures at 48 hr prior to transfection, 1 hr prior to transfection, 0.5 hr post-transfection, or 24 hrs post-transfection. Control cultures without momelotinib added were also examined. At 4 days post-transfection, cells were harvested and lysed and resulting lysates were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Graph displays exemplary data of genome titer (Y-axis) for various timings (hours (h)) of momelotinib addition in relation to transfection (X-axis).DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0039] Technologies of the present disclosure may be understood more readily by reference to the following detailed description of certain embodiments.Definitions

[0040] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

[0041] As used herein in the present disclosure, unless otherwise clear from context, (i) the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional / second one or more; (v) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included.

[0042] About: As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0043] Adeno-associated virus (AAV): As used herein, the terms “adeno-associated vims” andPage 18 of 10712963423v 1Attorney Docket No.: 2011256-2625“AAV” refer to viral particles, in whole or in part, of family Parvoviridae and genus Dependoparvovirus. AAV is a small replication-defective, nonenveloped virus. AAV may include, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAV serotype rhlO, AAV serotype rh74, AAV from the HSC 1-17 series, AAV from the CBr, CLv or CLg series, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any variant of any of the foregoing. AAV may also include engineered or chimeric versions of a wild-type AAV that include one or more insertions, deletions and / or substitutions within the Cap polypeptide(s) that affect one or more properties of the wildtype AAV serotype, including without limitation tropism and evasion of neutralizing antibodies (e.g., AAV- DJ, AAV-PHP.B, AAV-PHP.N, AAV.CAP-B1 to AAV.CAP-B25 and variants thereof). Wild-type AAV is reportedly replication deficient and requires co-infection of cells by a helper virus (e.g., adenovirus, herpes, or vaccinia virus) or supplementation of helper viral genes in order to replicate.

[0044] Agent: As used herein, the term “agent” refers to a compound or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively, or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and / or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them.

[0045] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal and / or a clone.

[0046] Cell density: As used herein, the term “cell density” refers to that number of cells present in a given volume of medium or that number of cells present in a given sur face area. For example, cell density may be represented as viable cells (vc) / ml or vc / cm2of culture medium.Page 19 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0047] Culture: As used herein, the terms “culture” and “cell culture” refer to a cell population (e.g., a eukaryotic cell population) that is suspended in or covered by a medium under conditions suitable to survival and / or growth of a cell population. As will be clear to those of ordinary skill in the art, these terms can also refer to a combination comprising a cell population and medium.

[0048] Derivative: As used herein, the term “derivative” refers to a structural analogue of a reference substance. That is, a “derivative” is a substance that shows significant structural similarity with the reference substance, such as sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, a derivative is a substance that can be generated from a reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be generated through performance of a synthetic process substantially similar' to (e.g., sharing a plurality of steps with) one that generates the reference substance.

[0049] Host cell: As used herein, the term “host cell” refers to a cell into which exogenous nucleic acid (recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Host cells can include prokaryotic and eukaryotic cells selected from any clade of life that are suitable for expressing an exogenous nucleic acid sequence (e.g., a recombinant nucleic acid sequence). Exemplary host cells include, but are not limited to, prokaryotes and eukaryotes (e.g., single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., or Streptomyces pp.). mycobacteria cells, fungal cells, yeast cells (e.g., .S’, cerevisiae, S. pombe, P. pastoris, or P. methanolica ). plant cells, insect cells (e.g. , Sf9, Sf21 , bacu lovi rus-infcctcd insect cells, or Trichoplusia ni), non-human animal cells, human cells, and cell fusions (e.g., hybridomas or quadromas). In some embodiments, a host cell comprises or is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, a host cell is a eukaryotic cell chosen from: a CHO (e.g., CHO-K, CHO-DXB1 1 , Veggie- CHO), COS (e.g., COS-7), retinal, Vero, CV1, kidney (e.g., HEK293 (e.g., HEK293T, HEK293F, HEK293FT, HEK293FTM, HEK293SG, HEK293H, HEK293E, HEK293A, or another variant of HEK293)), 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127, SP2 / 0, NS-0, MMT 060562, Sertoli, BRL 3 A cell, HT1080, myeloma cell, tumor cell, or a cell line derived from an aforementioned cell. Host cells used in methods, compositions, and systems described herein may comprise or be adherent or suspension cells.

[0050] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g.,Page 20 of 10712963423v 1Attorney Docket No.: 2011256-2625 animal, plant and / or microbe).

[0051] In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant and / or microbe).

[0052] Medium: As used herein, the terms “medium”, “culture medium”, and “growth medium” refer to a solution comprising nutrients to nourish cells (e.g., growing cells, e.g., mammalian cells). Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and / or trace elements required by cells for survival and / or minimal growth. Solutions can also comprise components that enhance survival and / or growth above a minimal rate, such as hormones and growth factors. Solutions can be formulated to a pH and concentration of one or more salts that are optimal for cellular survival and / or proliferation. For example, medium can be a “defined medium” or “chemically defined medium”, e.g., a serum-free medium that contains no proteins, hydrolysates, or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure. One of skill in the art understands defined media can comprise recombinant polypeptides including, but not limited to, hormones, cytokines, interleukins, and / or other signaling molecules.

[0053] Microtubule destabilizing agent: As used herein, the term “microtubule destabilizing agent” refers to any agent that interferes directly or indirectly with stability of microtubule polymers and / or microtubule polymerization, thereby inhibiting the physiological function of microtubules. Microtubules are polymers of tubulin and a major component of mitotic spindles, which separate duplicated chromosomes during cell division. If microtubules are destabilized, such as by a microtubule destabilizing agent, cells cannot divide into daughter cells. A microtubule destabilizing agent can reportedly arrest cell cycle of cells, such as by depolarizing microtubules, inactivating spindle fiber formation, blocking polymerization of tubules by binding tubulin, depolarizing microtubules by binding tubulin and inducing self-association, and / or inhibiting tubulin formation. In some embodiments, a microtubule destabilizing agent comprises or is a cell cycle arresting agent, e.g., a cell cycle arresting agent described herein. In some embodiments, a microtubule destabilizing agent comprises or is a G2 / M inhibitor. In some embodiments, a microtubule destabilizing agent comprises or is colcemid or a derivative or salt thereof. In some embodiments, a microtubule destabilizing agent comprises or is colchicine or a derivative or salt thereof. In some embodiments, a microtubule destabilizing agent comprises or is a Vinca alkaloid (e.g., vinblastine or vincristine or a derivative or salt thereof). In some embodiments, a microtubule destabilizing agent comprises or is vinblastine or a derivative or salt thereof. In some embodiments, a microtubule destabilizing agent comprises or is vincristine or a derivative or salt thereof. In some embodiments, a microtubule destabilizing agent comprises or is a microtubule destabilizing agent described in WO 2022 / 159702, which is herein incorporated by reference in its entirety.Page 21 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0054] Nucleic acid: The term “nucleic acid”, as used herein, includes any nucleotides and polymers thereof. The term “polynucleotide”, as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof. These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double - and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA comprising modified nucleotides and / or modified polynucleotides, such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotidic linkages. The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotidic linkages. In some embodiments, a "nucleic acid" is or comprises ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a -D-ribo configuration, a-LNA having an ot-L-ribo configuration (a diastereomer of LNA), 2’-amino-LNA having a 2’ -amino functionalization, and 2’-amino-a-LNA having a 2’ -amino functionalization), or a combination thereof. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a functional gene product such as a RNA, polypeptide, or protein.

[0055] One or more: As used herein, in some embodiments, “one or more” is 1-200, 1-150, 1-100, 1- 90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, “one or more” is one. In some embodiments, “one or more” is two. In some embodiments, “one or more” is three. In some embodiments, “one or more” is four. In some embodiments, “one or more” is five. In some embodiments, “one or more” is six. In some embodiments, “one or more” is seven. In some embodiments, “one or more” is eight. In some embodiments, “one or more” is nine. In some embodiments, “one or more” is ten. In some embodiments, “one or more” is at least one. In some embodiments, “one or more” is at least two. In some embodiments, “one or more” is at least three. In some embodiments, “one or more” is at least four. InPage 22 of 10712963423v 1Attorney Docket No.: 2011256-2625 some embodiments, “one or more” is at least five. In some embodiments, “one or more” is at least six. In some embodiments, “one or more” is at least seven. In some embodiments, “one or more” is at least eight. In some embodiments, “one or more” is at least nine. In some embodiments, “one or more” is at least ten.

[0056] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0057] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0058] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable earner” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable earners include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions;Page 23 of 10712963423v 1Attorney Docket No.: 2011256-2625 polyesters, polycarbonates and / or poly anhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0059] Polypeptide: As used herein, the term “polypeptide” refers to a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (e.g., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30% to about 40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, 99%, or greater in one or more highly conserved regions, usually encompassing at least 3 amino acids to 4 amino acids and often up to 20 amino acids or more, with another polypeptide of the same class, is encompassed within the term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both. Polypeptides may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, and / or methylation. In some embodiments, polypeptides comprise natural amino acids, non-natural amino acids, synthetic amino acids, or combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0060] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject is a human. In some embodiments, a subject may be suffering from and / or susceptible to a disease, disorder and / or condition.

[0061] Titer: As used herein, the term “titer” refers to a quantity of virus in a given volume. Titer, for example, can be expressed as viral genome copies (vg) per given volume or plaque forming units (pfu) per given volume. In some embodiments, titer can be expressed as number of capsids per given volume.

[0062] Transfection: As used herein, the term “transfection” refers to introduction of nucleic acid sequences, such as DNA or RNA (e.g., mRNA), into cells, such as eukaryotic cells (e.g., mammalian cells). For example, transfection can include vector-based transfection, viral-based transfection, electroporation, lipofection (e.g., with cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle- bascd transfection, and / or transfection based on cationic polymers (e.g., polycthylcniminc (PEI) or diethylaminoethyl-dextran (DEAE-dextran)).Page 24 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0063] Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked, e.g., into a cell (e.g., a host cell described herein), tissue, and / or organism. By way of non-limiting example, one type of vector is a “viral vector”, in which additional DNA segments may be ligated into a viral genome. Another type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to as “expression vectors.”

[0064] Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).Description of Certain Embodiments

[0065] Among other things, the present disclosure provides methods, systems, and compositions for production of viral vectors, e.g., recombinant viral vectors, e.g., recombinant adeno-associated viruses (rAAVs). In some embodiments, the present disclosure provides methods, systems, and compositions for producing a plurality of recombinant viral vector particles, e.g., rAAV particles.

[0066] The present disclosure is based, in part, on the discovery that the use of at least one of various additives (e.g., compounds, compositions thereof) described herein can provide increased titers of produced viral vectors, e.g., recombinant viral vectors, e.g., rAAVs. In contrast to production of proteins by transient transfection of host cells, which are normal cellular by-products, production of recombinant viral vector particles (e.g., rAAV particles) poses challenges in greater complexity due to the relationship between recombinant viral vectors and host cells. See, e.g., Walsh et al. (2013) Cold Spring Harb Perspect Biol. 2013 5(1), a012351, which is hereby incorporated by reference in its entirety. Recombinant viral vectors depend on cellular machinery for reproduction, while host cells have defense mechanisms against recombinant viral vectors as foreign particles. Further, recombinant viral vectors have mechanisms to inhibit host cell defenses. For example, Rep and Cap polypeptides can be toxic to host cells.

[0067] Despite these challenges, the present disclosure provides, among other things, a recombinant viral vector (e.g., rAAV) production process (e.g., a large-scale manufacturing process) using at least one additive described herein that efficiently generates high titers (e.g., genome titer) of recombinant viralPage 25 of 10712963423v 1Attorney Docket No.: 2011256-2625 vector particles (e.g., rAAV particles). In some embodiments, a higher titer of recombinant viral vector particles (e.g., rAAV particles) is relative to recombinant viral vector particles produced under the same conditions and in the same medium, but without the at least one additive. In some embodiments, an additive is momelotinib or a salt or derivative thereof.Additives

[0068] Among other things, the present disclosure provides various additives (e.g., compounds, compositions thereof) that are added to a medium (e.g., a culture medium, a growth medium) to modulate production of viral vectors, e.g., recombinant viral vectors, e.g., rAAVs.

[0069] In some embodiments, an additive can provide increased titer of produced recombinant viral vector particles when added to a medium as compared to a titer achieved with a reference medium, e.g., a medium comprising the additive. In some embodiments, an additive can provide increased titer of a produced recombinant viral vector particles when added to a medium as compared to a titer achieved with a reference medium, e.g., a medium not comprising the additive. In some embodiments, a first additive can provide increased titer of a produced recombinant viral vector particles when added to a medium as compared to a titer achieved with a reference medium, e.g., a medium comprising a second additive and not comprising the first additive.

[0070] In some embodiments, an additive comprises or is a Janus kinase (JAK) inhibitor. In some embodiments, a JAK inhibitor can provide inhibition of JAK1, JAK2, JAK3, and / or TYK2. In some embodiments, an additive comprises or is a JAK1 inhibitor. In some embodiments, an additive comprises or is a JAK2 inhibitor. In some embodiments, an additive comprises or is a JAK1 and JAK2 inhibitor. In some embodiments, an additive comprises or is a JAK3 inhibitor, hr some embodiments, an additive comprises or is a TYK2 inhibitor.

[0071] In some embodiments, an additive comprises or is momelotinib or a salt or derivative thereof. In some embodiments, a JAK inhibitor comprises or is momelotinib or a salt or derivative thereof. In some embodiments, a JAK1 inhibitor comprises or is momelotinib or a salt or derivative thereof. In some embodiments, a JAK2 inhibitor comprises or is momelotinib or a salt or derivative thereof. In some embodiments, a salt of momelotinib comprises or is momelotinib dihydrochloride. Momelotinib may also be referred to as N-(cyanomethyl)-4-[2-(4-morpholin-4-ylanilino)pyrimidin-4-yl]benzamide, CYT387, CYT-11387, or GS-0387. Momelotinib reportedly acts as a small molecule inhibitor of JAK1 and JAK2 by competing with binding of ATP to the aforementioned kinases. See, e.g., Pardanani et al. Leukemia. 2009;23(8): 1441-1445., which is hereby incoiporated by reference in its entirety. Momelotinib may have a structure as depicted in the following general formula:Page 26 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0072] In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM to about 25 pM, about 0.25 |1M to about 20 |1M, about 0.25 pM to about 15 |JM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, about 1 pM to about 2 pM, about 1.5 pM to about 25 pM, about 1.5 pM to about 20 pM, about 1.5 pM to about 15 pM, about 1.5 pM to about 10 pM, about 1.5 pM to about 5 pM, about 1.5 pM to about 2.5 pM, or about 1.5 pM to about 2 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM or more. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.1 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.2 pM. hr some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.3 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.4 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.6 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.7 pM. hr Page 27 of 10712963423v 1Attorney Docket No.: 2011256-2625 some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.8 p M. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 1.9 M. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 2 |1M. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 2.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 3 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about3.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 4 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 4.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 5.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 6 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about6.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 7 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 7.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 8 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 8.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 9 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about9.5 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 10 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 11 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 12 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 13 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 14 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 15 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 16 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 17 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 18 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 19 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 20 pM. InPage 28 of 10712963423v 1Attorney Docket No.: 2011256-2625 some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 21 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 22 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 23 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 24 pM. In some embodiments, momelotinib or a salt or derivative thereof is added to a medium to a concentration of about 25 pM.

[0073] In some embodiments, an additive comprises or is ruxolitinib or a salt or derivative thereof. In some embodiments, a JAK inhibitor comprises or is ruxolitinib or a salt or derivative thereof. In some embodiments, a JAK1 inhibitor comprises or is ruxolitinib or a salt or derivative thereof. In some embodiments, a JAK2 inhibitor comprises or is ruxolitinib or a salt or derivative thereof. In some embodiments, a salt of ruxolitinib comprises or is ruxolitinib phosphate. Ruxolitinib may also be referred to as (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-l-ylJpropanenitrile, INCB018424, or INC424. Ruxolitinib may have a structure as depicted in the following general formula:

[0074] In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about2.5 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 2 pM to about 20 pM, about 2 pM to about 15 pM, about 2 pM to about 10 pM, or about 2 pM to about 5 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, or about 20 pM or more. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of aboutPage 29 of 10712963423v 1Attorney Docket No.: 2011256-26250.5 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 1 M. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 2 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 2.5 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 3 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 4 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 5 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 6 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 7 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 8 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 9 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 10 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 11 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 12 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 13 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 14 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 15 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 16 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 17 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 18 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 19 pM. In some embodiments, ruxolitinib or a salt or derivative thereof is added to a medium to a concentration of about 20 pM.

[0075] In some embodiments, an additive comprises or is a phosphoinositide-dependent kinase- 1 (PDKl)Zprotein kinase B (AKT) signaling inhibitor. In some embodiments, an additive comprises or is a TANK-binding kinase 1 (TBK1 ) inhibitor.

[0076] In some embodiments, an additive comprises or is BX-795 or a salt or derivative thereof. In some embodiments, a PDK1 / AKT signaling inhibitor comprises or is BX-795 or a salt or derivative thereof. In some embodiments, a TBK1 inhibitor comprises or is BX-795 or a salt or derivative thereof. In some embodiments, a salt of BX-795 comprises or is BX-795 hydrochloride. BX-795 may also be referred to asPage 30 of 10712963423v 1Attorney Docket No.: 2011256-2625BX795, BX 795, or N-[3-[[5-iodo-4-[3-(thiophene-2-carbonylamino)propylamino]pyrimidin-2- yl]amino]phenyl]pyrrolidine-l-carboxamide. BX-795 is reportedly a small molecule inhibitor of PDK1 that has been investigated for usage as an anti-tumor agent. See, e.g., Feldman et al. J Biol Chem. 2005;280(20):19867-19874., which is hereby incorporated by reference in its entirety. Further reports have detailed that BX-795 can inhibit TBK1 and IKKE, which may both be involved in modulation of NF-KB activation and corresponding downstream NF-KB -dependent gene transcription. See, e.g., Bain et al. Biochem J. 2007;408(3):297-315. and Clark et al. J Biol Chem. 2009;284(21): 14136-14146., which are hereby incorporated by reference in their entirety. BX-795 may have a structure as depicted in the following general formula:

[0077] In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM to about 10, about 0.1 pM to about 7.5, about 0.1 pM to about 5, about 0.1 pM to about 4, about 0.1 pM to about 3, about 0.1 pM to about 2, about 0.1 pM to about 1, about 0.25 pM to about 10, about 0.25 pM to about 7.5, about 0.25 pM to about 5, about 0.25 pM to about 4, about 0.25 pM to about 3, about 0.25 pM to about 2, about 0.25 pM to about 1, about 0.5 pM to about 10, about 0.5 pM to about 7.5, about 0.5 pM to about 5, about 0.5 pM to about 4, about 0.5 pM to about 3, about 0.5 pM to about 2, about 0.5 pM to about 1, about 0.75 pM to about 10, about 0.75 pM to about 7.5, about 0.75 pM to about 5, about 0.75 pM to about 4, about 0.75 pM to about 3, about 0.75 pM to about 2, about 0.75 pM to about 1 1 pM to about 10, about 1 pM to about 7.5 1 pM to about 5, about 1 pM to about 4, about 1 pM to about 3, or about 1 pM to about 2 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or about 10 pM or more. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM. In some embodiments, BX- 795 or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 0.5Page 31 of 10712963423v 1Attorney Docket No.: 2011256-2625 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1.1 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1.2 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1.3 pM. In some embodiments, BX- 795 or a salt or derivative thereof is added to a medium to a concentration of about 1.4 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1.6 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1.7 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1.8 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 1.9 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 2 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 2.5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 3 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 3.5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 4 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 4.5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 5.5 pM. In some embodiments, BX- 795 or a salt or derivative thereof is added to a medium to a concentration of about 6 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 6.5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 7 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 7.5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 8 pM. hr some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 8.5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 9 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 9.5 pM. In some embodiments, BX-795 or a salt or derivative thereof is added to a medium to a concentration of about 10 pM.

[0078] In some embodiments, an additive comprises or is an interferon (IFN) inhibitor. In somePage 32 of 10712963423v 1Attorney Docket No.: 2011256-2625 embodiments, an additive comprises or is a type I IFN inhibitor. In some embodiments, an additive comprises or is an IFN-a inhibitor. In some embodiments, an additive comprises or is an interferon-a- receptor (IFN-a-R) inhibitor. In some embodiments, an additive comprises or is an IFN-a and IFN-a-R inhibitor.

[0079] In some embodiments, an additive comprises or is interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof. In some embodiments, an IFN inhibitor comprises or is interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof. In some embodiments, a type I IFN inhibitor comprises or is interferon-a-IFNa-R interaction inhibitor a salt or derivative thereof. In some embodiments, an IFN- a inhibitor comprises or is interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof. In some embodiments, an IFN-a-R inhibitor comprises or is interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof. In some embodiments, a salt of interferon-a-IFNa-R interaction inhibitor comprises or is interferon-a-IFNa-R interaction inhibitor hydrochloride. Interferon-a-IFNa-R interaction inhibitor may also be referred to as N-methyl-l-(2-naphthalen-l-ylsulfanylphenyl)methanamine. Interferon-a-IFNa-R interaction inhibitor reportedly binds IFN-a and thus blocks the interaction of IFN-a with IFNa-R. See, e.g., Geppert et al. Angew Chem Int Ed Engl. 2012;51(l):258-261., which is hereby incorporated by reference in its entirety. Interferon-a-IFNa-R interaction inhibitor may have a structure as depicted in the following general formula:

[0080] In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM to about 25 pM, about 0.1 pM to about 20 pM, about 0.1 pM to about 15 pM, about 0.1 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 2.5 pM, about 0.1 pM to about 1 pM, about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, about 1 pM to about 2 pM, about 1.5 pM to about 25 pM, about 1.5 pM to about 20 pM, about 1.5 pM to about 15 pM, about 1.5 pM to about 10 pM, about 1.5 pM to about 5 pM, about 1.5 pM to about 2.5 pM, or about 1.5 pM to about 2 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM, about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1Page 33 of 10712963423v 1Attorney Docket No.: 2011256-2625 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM or more. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.1 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.2 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.3 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.4 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.6 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.7 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.8 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.9 pM. hr some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 2 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 2.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 3 pM. In some embodiments, intcrfcron-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 3.5 pM. In some embodiments,Page 34 of 10712963423v 1Attorney Docket No.: 2011256-2625 interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 4 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 4.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 5.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 6 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 6.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 7 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 7.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 8 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 8.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 9 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 9.5 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 10 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 11 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 12 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 13 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 14 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 15 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 16 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 17 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 18 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 19 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to aPage 35 of 10712963423v 1Attorney Docket No.: 2011256-2625 concentration of about 20 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 21 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 22 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 23 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 24 pM. In some embodiments, interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 25 pM.

[0081] In some embodiments, an additive comprises or is an IKB kinase (IKK) inhibitor. In some embodiments, an additive comprises or is an IKK-1 inhibitor. In some embodiments, an additive comprises or is an IKK-2 inhibitor. In some embodiments, an additive comprises or is an IKK-1 and IKK-2 inhibitor. IKK reportedly acts to phosphorylate IKB proteins, e.g., ItcBa proteins to lead to dissociation of ItcBa from NF-KB transcription factor and subsequent transcription of NF-KB -dependent genes, e.g., immune and inflammatory response genes. See, e.g., Hinz and Scheidereit. EMBO Rep. 2014; 15(1):46-61. , which is hereby incorporated by reference in its entirety.

[0082] In some embodiments, an additive comprises or is BMS-345541 or a salt or derivative thereof. In some embodiments, an IKK inhibitor comprises or is BMS-345541 or a salt or derivative thereof. In some embodiments, an IKK-1 inhibitor comprises or is BMS-345541 or a salt or derivative thereof. In some embodiments, an IKK-2 inhibitor comprises or is BMS-345541 or a salt or derivative thereof. In some embodiments, BMS-345541 comprises or is a free base form of BMS-345541. In some embodiments, a salt of BMS-345541 comprises or is BMS-345541 hydrochloride. BMS-345541 may also be referred to as N'-(l,8-dimethylimidazo[l,2-a]quinoxalin-4-yl)ethane-l,2-diamine. BMS-345541 is reportedly a small molecule that can act as a selective inhibitor of the catalytic subunits of IKK, i.e., IKK-1 and IKK-2. Accordingly, BMS-345541 may reportedly be used to inhibit activation of NF-KB and transcription of downstream inflammatory response genes. See, e.g., Burke et al. J Biol Chem. 2003;278(3): 1450-1456., which is hereby incorporated by reference in its entirety. BMS-345541 may have a structure as depicted in the following general formula:Page 36 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0083] In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM to about 25 pM, about 0.1 pM to about 20 pM, about 0.1 pM to about 15 pM, about 0.1 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 2.5 pM, about 0.1 pM to about 1 pM, about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, about 1 pM to about 2 pM, about 1.5 pM to about 25 pM, about 1.5 pM to about 20 pM, about 1.5 pM to about 15 pM, about 1.5 pM to about 10 pM, about 1.5 pM to about 5 pM, about 1.5 pM to about 2.5 pM, or about 1.5 pM to about 2 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM, about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM or more. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, BMS- 345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 1 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 1.1 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 1.2 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 1.3 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 1.4 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 1.6 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 1.7 pM. In some embodiments, BMS-345541 or a salt orPage 37 of 10712963423v 1Attorney Docket No.: 2011256-2625 derivative thereof is added to a medium to a concentration of about 1.8 pM. In some embodiments, BMS- 345541 or a salt or derivative thereof is added to a medium to a concentration of about 1.9 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 2 M. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 2.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 3 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 3.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 4 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 4.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 5.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 6 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 6.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 7 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 7.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 8 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 8.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 9 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 9.5 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 10 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 11 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 12 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 13 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 14 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 15 pM. In some embodiments, BMS- 345541 or a salt or derivative thereof is added to a medium to a concentration of about 16 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 17 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 18 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 19 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 20 pM. In some embodiments, BMS-345541 orPage 38 of 10712963423v 1Attorney Docket No.: 2011256-2625 a salt or derivative thereof is added to a medium to a concentration of about 21 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 22 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 23 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 24 pM. In some embodiments, BMS-345541 or a salt or derivative thereof is added to a medium to a concentration of about 25 pM.

[0084] In some embodiments, an additive comprises or is TPCA-1 or a salt or derivative thereof. In some embodiments, an IKK inhibitor comprises or is TPCA-1 or a salt or derivative thereof. In some embodiments, an IKK-2 inhibitor comprises or is TPCA-1 or a salt or derivative thereof. In some embodiments, an additive comprises or is a NF-KB inhibitor. In some embodiments, a NF-KB inhibitor comprises or is TPCA-1. TPCA-1 reportedly acts as a selective inhibitor of IKK-2 and may also act as a direct inhibitor of STAT3. See, e.g., Podolin et al. J Pharmacol Exp Ther. 2005;312( 1 ):373-381. and Nan et al. Mol Cancer Ther. 2014;13(3):617-629., which are hereby incorporated by reference in their entirety. TPCA-1 may also be referred to as IKK Inhibitor IV, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3- thiophenecarboxamide, or 5-(4-Fluorophenyl)-2-ureidothiophene-3-carboxamide. TPCA-1 may have a structure as depicted in the following general formula:

[0085] In some embodiments, an additive comprises or is IMD 0354 or a salt or derivative thereof. In some embodiments, an IKK inhibitor comprises or is IMD 0354 or a salt or derivative thereof. In some embodiments, a NF-KB inhibitor comprises or is IMD 0354. IMD 0354 can reportedly act as an inhibitor of NF-KB though binding with IKK, which is involved in the modulation of NF-KB activity. See, e.g., Tanaka et al. Blood. 2005; 105(6):2324-2331., which is hereby incorporated by reference in its entirety. IMD 0354 may also be referred to as N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide. IMD 0354 may have a structure as depicted in the following general formula:Page 39 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0086] In some embodiments, an additive comprises or is BAY 11-7082 or a salt or derivative thereof. In some embodiments, an IKK inhibitor comprises or is BAY 11-7082 or a salt or derivative thereof. In some embodiments, a NF-KB inhibitor comprises or is BAY 11-7082. BAY 11-7082 may also be referred to as (E)-3-tosylacrylonitrile or (E)-3-(4-methylphenyl)sulfonylprop-2-enenitrile. BAY 11-7082 may have a structure as depicted in the following general formula:

[0087] In some embodiments, an additive comprises or is an inhibitor of endonuclease-mediated DNA fragmentation. In some embodiments, an additive comprises or is an inhibitor of IFN-gamma.

[0088] In some embodiments, an additive comprises a polyamine. In some embodiments, a polyamine comprises or is spermine. In some embodiments, an additive comprises or is spermine. In some embodiments, an inhibitor of endonuclease-mediated DNA fragmentation comprises or is spermine. In some embodiments, an inhibitor of IFN-gamma comprises or is spermine. Spermine may also be referred to as N,N'-bis(3-aminopropyl)butane-l,4-diamine. Spermine may have a structure as depicted in the following general formula:

[0089] In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM to about 250 pM, about 0.5 pM to about 200 pM, about 0.5 pM to about 150 pM, about 0.5 pM to about 100 pM, about 0.5 pM to about 75 pM, about 0.5 pM to about 50 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 75 pM, about 1 pM to about 50 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2 pM to about 150 pM, about 2 pM to about 100 pM, about 2 pM to about 75 pM, about 2 pM to about 50 pM, about 2 pM to about 25 pM, about 2 pM to about 20 pM, about 2 pM to about 15 pM, about 2 pM to about 10 pM, about 2 pM to about 5 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 75 pM, about 5 pM to about 50 pM, about 5 pM to about 25 pM, about 5 pM to about 20 pM, about 5 pM to about 15 pM, about 5 pM to about 10 pM, about 10 pM to about 250 pM, about 10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about 100 pM, about 10 pM to about 75 pM, about 10 pM to about 50 pM, about 10 pM to about 25 pM, about 10Page 40 of 10712963423v 1Attorney Docket No.: 2011256-2625 pM to about 20 pM, about 25 pM to about 250 pM, about 25 pM to about 200 pM, about 25 pM to about 150 pM, about 25 pM to about 100 pM, about 25 pM to about 75 pM, about 25 pM to about 50 pM, about 50 pM to about 250 pM, about 50 pM to about 200 pM, about 50 pM to about 150 pM, about 50 pM to about 100 pM, about 50 pM to about 75 pM, about 75 pM to about 250 pM, about 75 pM to about 200 pM, about 75 pM to about 150 pM, about 75 pM to about 100 pM, about 100 pM to about 250 pM, about 100 pM to about 200 pM, or about 100 pM to about 150 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 50 pM, about 75 pM, about 100 pM, about 150 pM, about 200 pM, or about 250 pM or more. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 1 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 2 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 2.5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 3 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 3.5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 4 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 4.5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 5.5 pM. Tn some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 6 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 6.5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 7 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 7.5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 8 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 8.5 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 9 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 9.5 pM. In somePage 41 of 10712963423v 1Attorney Docket No.: 2011256-2625 embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 10 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 11 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 12 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 13 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 14 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 15 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 16 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 17 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 18 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 19 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 20 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 21 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 22 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 23 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 24 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 25 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 50 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 75 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 100 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 150 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 200 pM. In some embodiments, spermine or a salt or derivative thereof is added to a medium to a concentration of about 250 pM.

[0090] In some embodiments, an additive comprises or is a MNK1 inhibitor. In some embodiments, an additive comprises or is CGP 57380 or a salt or derivative thereof. In some embodiments, a MNK1 inhibitor comprises or is CGP 57380 or a salt or derivative thereof. See, e.g., Knauf et al. Mol Cell Biol. 2001 ;21 ( 16):5500-5511., which is hereby incorporated by reference in its entirety. CGP 57380 may also be referred to as N3-(4-fluorophenyl)-lh-pyrazolo[3,4-d]pyrimidine-3,4-diamine. CGP 57380 may have a structure as depicted in the following general formula:Page 42 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0091] In some embodiments, an additive comprises or is a proteasome inhibitor. In some embodiments, an additive comprises or is epoxomicin or a salt or derivative thereof. In some embodiments, a proteasome inhibitor comprises or is epoxomicin or a salt or derivative thereof. Epoxomicin reportedly acts as a proteasome inhibitor that exhibits anti-tumor activity. See, e.g., Hanada et al. J Antibiot (Tokyo). 1992;45(11):1746-1752. and Meng et al. Proc Natl Acad Sci USA. 1999;96(18):10403-10408., which are hereby incorporated by reference in their entirety. Epoxomicin may also be referred to as (2S,3S)-2- [[(2S,3S)-2-[acetyl(niethyl)amino]-3-methylpentanoyl]amino]-N-[(2S,3R)-3-hydroxy-l-[[(2S)-4-methyl- 1 -[(2R)-2-methyloxiran-2-yl] - 1 -oxopentan-2-yl]amino]- 1 -oxobutan-2-yl] -3-methylpentanamide.Epoxomicin may have a structure as depicted in the following general formula:

[0092] In some embodiments, an additive comprises or is lactacystin or a salt or derivative thereof. In some embodiments, a proteasome inhibitor comprises or is lactacystin or a salt or derivative thereof. Lactacystin reportedly is a microbial compound that inhibits the 20S proteasome. See, e.g., Omura et al. J Antibiot (Tokyo). 1991 ;44(1): 113-116. and Fenteany et al. Science. 1995;268(5211):726-731., which are hereby incorporated by reference in their entirety. Lactacystin may also be referred to as (2R)-2-acetamido- 3-[(2R,3S,4R)-3-hydroxy-2-[(lS)-l-hydroxy-2-methylpropyl]-4-methyl-5-oxopyrrolidine-2- carbonyl] sulfanylpropanoic acid. Lactacystin may have a structure as depicted in the following general formula:

[0093] In some embodiments, an additive comprises or is a protein neddylation inhibitor. In some embodiments, an additive comprises or is MLN-4924 or a salt or derivative thereof. In some embodiments, Page 43 of 10712963423v 1Attorney Docket No.: 2011256-2625 a protein neddylation inhibitor comprises or is MLN-4924 or a salt or derivative thereof. MLN-4924 reportedly acts as an inhibitor of NEDD8-activating enzyme (NAE). See, e.g., Soucy et al. Nature. 2009;458(7239):732-736., which is hereby incorporated by reference in its entirety. MLN-4924 may also be referred to as MLN4924, pevonedistat, or [(lS,2S,4R)-4-[4-[[(lS)-2,3-dihydro-lH-inden-l- yl]amino]pyrrolo[2,3-d]pyrimidin-7-yl]-2-hydroxycyclopentyl]methyl sulfamate. MLN-4924 may have a structure as depicted in the following general formula:

[0094] In some embodiments, an additive comprises or is NF-KB activation inhibitor or a salt or derivative thereof. NF-KB activation inhibitor reportedly inhibits NF-KB activation by blocking I K K|3. See, e.g., Kim ct al. Mol Pharmacol. 2008;73(4):1309-1318., which is hereby incorporated by reference in its entirety. NF-KB activation inhibitor may also be referred to as NF-KB activation inhibitor VI, BOT-64, or 6,6-dimethyl-2-phenylimino-5,7-dihydro-l,3-benzoxathiol-4-one. NF-KB activation inhibitor may have a structure as depicted in the following general formula:

[0095] In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM to about 25 pM, about 0.1 pM to about 20 pM, about 0.1 pM to about 15 pM, about 0.1 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 2.5 pM, about 0.1 pM to about 1 pM, about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, about 1 pM to about 2 pM, about 1.5 pM to about 25 pM, about 1.5 pM to about 20 pM, about 1.5 pM to about 15 pM, about 1.5 pM to about 10 pM, about 1.5 pM to about 5 pM, about 1.5 pM to about 2.5 pM, or about 1.5 pM to about 2 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of aboutPage 44 of 10712963423v 1Attorney Docket No.: 2011256-26250.05 pM, about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.25 pM, about 1.5 pM, about 1.75 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM or more. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.25 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 1.75 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 2 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 2.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 3 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 3.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 4 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 4.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 5.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 6 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 6.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 7 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 7.5 pM. In some embodiments, NF-KB activationPage 45 of 10712963423v 1Attorney Docket No.: 2011256-2625 inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 8 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 8.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 9 ,u M. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 9.5 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 10 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentr ation of about 11 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 12 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 13 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 14 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 15 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 16 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 17 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 18 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 19 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentr ation of about 20 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 21 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 22 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 23 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 24 pM. In some embodiments, NF-KB activation inhibitor or a salt or derivative thereof is added to a medium to a concentration of about 25 pM.

[0096] In some embodiments, an additive comprises or is parthenolide or a salt or derivative thereof. Parthenolide reportedly inhibits NF-KB activation and thus inhibit a variety of cytokines. See, e.g., Zhu et al. Front Pharmacol. 2023; 14: 1111218., which is hereby incorporated by reference. Parthenolide may also be referred to as PTL, PAR, or (lS,2R,4R,7E,HS)-4,8-dimethyl-12-methylidene-3,14- dioxatricyclo[9.3.0.02,4]tetradec-7-en-13-one. Parthenolide may have a structure as depicted in the following general formula:Page 46 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0097] In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about2.5 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 2 pM to about 20 pM, about 2 pM to about 15 pM, about 2 pM to about 10 pM, or about 2 pM to about 5 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM. about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, or about 20 pM or more. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 1 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 2 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 2.5 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 3 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 4 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 5 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 6 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 7 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 8 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 9 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 10 pM. In some embodiments, parthenolide or a salt or derivative thereof is addedPage 47 of 10712963423v 1Attorney Docket No.: 2011256-2625 to a medium to a concentration of about 11 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 12 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 13 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 14 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 15 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 16 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 17 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 18 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 19 pM. In some embodiments, parthenolide or a salt or derivative thereof is added to a medium to a concentration of about 20 pM.

[0098] In some embodiments, an additive comprises or is a histone deacetylase (HD AC) inhibitor. In some embodiments, an additive comprises or is trichostatin A or a salt or derivative thereof. In some embodiments, a HDAC inhibitor comprises or is trichostatin A or a salt or derivative thereof. Trichostatin A reportedly acts as an inhibitor of class I and class II HDACs and can provide anti-inflammatory activities through multiple mechanisms. See, e.g., Bouyahya et al. Pharmaceuticals (Basel). 2022; 15(10): 1235., which hereby is incorporated by reference in its entirety. Trichostatin A may also be referred to as TSA or (2E,4E,6R)-7-|4-(dimethylamino)phenyl|-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide.Trichostatin A may have a structure as depicted in the following general formula:

[0099] In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 0.01 pM to about 2.0 pM, about 0.01 pM to about 1.75 pM, about 0.01 pM to about 1.5 pM, about 0.01 pM to about 1.25 pM, about 0.01 pM to about 1.0 pM, about 0.01 pM to about 0.75 pM, about 0.01 pM to about 0.5 pM, about 0.01 pM to about 0.25 pM, about 0.01 pM to about 0.1 pM, about 0.01 pM to about 0.05 pM, about 0.05 pM to about 2.0 pM, about 0.05 pM to about 1.75 pM, about 0.05 pM to about 1.5 pM, about 0.05 pM to about 1.25 pM, about 0.05 pM to about 1.0 pM, about 0.05 pM to about 0.75 pM, about 0.05 pM to about 0.5 pM, about 0.05 to about 0.25 pM, about 0.05 to about 0.1 pM, about 0.1 pM to about 2.0 pM, about 0.1 pM to about 1.75 pM, about 0.1 pM to about 1.5 pM, about 0.1 pM to about 1.25 pM, about 0.1 pM to about 1.0 pM, about 0.1 pM to about 0.75 pM, about 0.1 pM to about 0.5 pM, or about 0.1 pM to about 0.25 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 0.01 pM, about 0.05 pM, about 0.1 pM,Page 48 of 10712963423v 1Attorney Docket No.: 2011256-2625 about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1.0 pM, about 1.25 pM, about 1.5 pM, about 1.75 pM, or about 2.0 pM or more. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 0.01 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 1.0 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 1.25 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 1.75 pM. In some embodiments, trichostatin A or a salt or derivative thereof is added to a medium to a concentration of about 2.0 pM.

[0100] In some embodiments, an additive comprises or is a protein kinase R (PKR) inhibitor. In some embodiments, an additive comprises or is imidazolo-oxindole or a salt or derivative thereof. In some embodiments, a PKR inhibitor comprises or is imidazolo-oxindole or a salt or derivative thereof. Imidazolo-oxindole reportedly acts as an inhibitor of PKR, which is a protein activated by dsRNA that modulates cellular responses to viral infection. See, e.g., Ingrand et al. FEBS Lett. 2007;581(23):4473- 4478., which is hereby incorporated by reference in its entirety. Imidazolo-oxindole may also be referred to as imidazolo-oxindole PKR inhibitor C16, PKR inhibitor, or (8Z)-8-(lH-imidazol-5-ylmethylidene)-6H- pyrrolo[2,3-g][l,3]benzothiazol-7-one. Imidazolo-oxindole may have a structure as depicted in the following general formula:

[0101] In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 0.01 pM to about 2.0 pM, about 0.01 pM to about 1.75 pM, about 0.01 pM to about 1.5 pM, about 0.01 pM to about 1.25 pM, about 0.01 pM to about 1.0 pM, about 0.01 pM to about 0.75 pM, about 0.01 pM to about 0.5 pM, about 0.01 pM to about 0.25 pM, about 0.01 pM to about 0.1 pM, about 0.01 pM to about 0.05 pM, about 0.05 pM to about 2.0 pM, about 0.05 pM to about 1.75 pM, about 0.05 pM to about 1.5 pM, about 0.05 pM to about 1.25 pM, about 0.05 pM to about 1.0 pM, aboutPage 49 of 10712963423v 1Attorney Docket No.: 2011256-26250.05 pM to about 0.75 pM, about 0.05 |aM to about 0.5 pM, about 0.05 to about 0.25 JJM, about 0.05 to about 0.1 pM, about 0.1 pM to about 2.0 pM, about 0.1 pM to about 1.75 pM, about 0.1 pM to about 1.5 pM, about 0.1 pM to about 1.25 pM, about 0.1 pM to about 1.0 pM, about 0.1 pM to about 0.75 pM, about 0.1 pM to about 0.5 pM, or about 0.1 pM to about 0.25 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1.0 pM, about 1.25 pM, about 1.5 pM, about 1.75 pM, or about 2.0 pM or more. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 0.01 pM. In some embodiments, imidazolo- oxindole or a salt or derivative thereof is added to a medium to a concentration of about 0.05 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 0.25 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 0.75 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 1.0 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 1.25 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 1.5 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 1.75 pM. In some embodiments, imidazolo-oxindole or a salt or derivative thereof is added to a medium to a concentration of about 2.0 pM.

[0102] In some embodiments, an additive comprises or is a tosylate or a salt or derivative thereof. In some embodiments, an additive comprises or is a tosylate salt or derivative thereof. Tosylate may also be referred to as 4-methylbenzenesulfonate or 4-toluene sulfonate. Tosylate may have a structure as depicted in the following general formula:

[0103] In some embodiments, tosylate salt or a derivate thereof is added to a medium to a concentration of about 0.01 pM to about 1.5 pM, about 0.01 pM to about 1.4 pM, about 0.01 pM to about 1.3 pM, about 0.01 pM to about 1.2 pM, about 0.01 pM to about 1.1 pM, about 0.01 pM to about 1.0 pM, about 0.01 pM to about 0.9 pM, about 0.01 pM to about 0.8 pM, about 0.01 pM to about 0.7 pM, about 0.01 pM to about 0.6 pM, about 0.01 pM to about 0.5 pM, about 0.01 pM to about 0.4 pM, about 0.01 pM to about 0.3 pM, about 0.01 pM to about 0.2 pM, about 0.01 pM to about 0.1 pM, about 0.01 pM to about 0.05 pM, aboutPage 50 of 10712963423v 1Attorney Docket No.: 2011256-26250.05 pM to about 1.5 pM, about 0.05 pM to about 1.4 pM, about 0.05 pM to about 1.3 pM, about 0.05 pM to about 1.2 pM, about 0.05 pM to about 1.1 pM, about 0.05 pM to about 1.0 pM, about 0.05 pM to about 0.9 pM, about 0.05 pM to about 0.8 pM, about 0.05 pM to about 0.7 pM, about 0.05 pM to about 0.6 pM, about 0.05 pM to about 0.5 pM, about 0.05 pM to about 0.4 pM, about 0.05 pM to about 0.3 pM, about 0.05 pM to about 0.2 pM, about 0.05 pM to about 0.1 pM, about 0.1 pM to about 1.5 pM, about 0.1 pM to about 1.4 pM, about 0.1 pM to about 1.3 pM, about 0.1 pM to about 1.2 pM, about 0.1 pM to about 1.1 pM, about 0.1 pM to about 1.0 pM, about 0.1 pM to about 0.9 pM, about 0.1 pM to about 0.8 pM, about 0.1 pM to about 0.7 pM, about 0.1 pM to about 0.6 pM, about 0.1 pM to about 0.5 pM, about 0.1 pM to about 0.4 pM, about 0.1 pM to about 0.3 pM, or about 0.1 pM to about 0.2 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.01 pM, about 0.025 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1.0 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM. about 1.4 pM, or about 1.5 pM or more. In some embodiments. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.01 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.025 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.05 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.075 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.1 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.2 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.3 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.4 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.5 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.6 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.7 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.8 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 0.9 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 1.0 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 1.1 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 1.2 pM. In some embodiments, tosylate salt or a derivative thereof is added to a medium to a concentration of about 1.3 pM. In some embodiments, tosylatc salt or a derivative thereof is added to a medium to a concentration of about 1.4 pM. In some embodiments, tosylate salt or a derivativePage 51 of 10712963423v 1Attorney Docket No.: 2011256-2625 thereof is added to a medium to a concentration of or about 1.5 pM or more.

[0104] In some embodiments, an additive described herein is added to a medium to a concentration of about 0.1 pM to about 250 pM, about 0.1 pM to about 225 pM, about 0.1 pM to about 200 pM, about 0.1 pM to about 175 pM, about 0.1 pM to about 150 pM, about 0.1 pM to about 125 pM, about 0.1 pM to about 100 pM, about 0.1 pM to about 90 pM, about 0.1 pM to about 80 pM, about 0.1 pM to about 70 pM, about 0.1 pM to about 60 pM, about 0.1 pM to about 50 pM, about 0.1 pM to about 40 pM, about 0.1 pM to about 30 pM, about 0.1 pM to about 25 pM, about 0.1 pM to about 20 pM, about 0.1 pM to about 15 pM, about 0.1 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 2.5 pM, about 0.1 pM to about 1 pM, about 0.25 pM to about 250 pM, about 0.25 pM to about 225 pM, about 0.25 pM to about 200 pM, about 0.25 pM to about 175 pM, about 0.25 pM to about 150 pM, about 0.25 pM to about 125 pM, about 0.25 pM to about 100 pM, about 0.25 pM to about 90 pM, about 0.25 pM to about 80 pM, about 0.25 pM to about 70 pM, about 0.25 pM to about 60 pM, about 0.25 pM to about 50 pM, about 0.25 pM to about 40 pM, about 0.25 pM to about 30 pM, about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 250 pM, about 0.5 pM to about 225 pM, about 0.5 pM to about 200 pM, about 0.5 pM to about 175 pM, about 0.5 pM to about 150 pM, about 0.5 pM to about 125 pM, about 0.5 pM to about 100 pM, about 0.5 pM to about 90 pM, about 0.5 pM to about 80 pM, about 0.5 pM to about 70 pM, about 0.5 pM to about 60 pM, about 0.5 pM to about 50 pM, about 0.5 pM to about 40 pM, about 0.5 pM to about 30 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 250 pM, about 1 pM to about 225 pM, about 1 pM to about 200 pM, about 1 pM to about 175 pM, about 1 pM to about 150 pM, about 1 pM to about 125 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, about 1 pM to about 2 pM, about 1.5 pM to about 250 pM, about 1.5 pM to about 225 pM, about 1.5 pM to about 200 pM, about 1.5 pM to about 175 pM, about 1.5 pM to about 150 pM, about 1.5 pM to about 125 pM, about 1.5 pM to about 100 pM, about 1.5 pM to about 90 pM, about 1.5 pM to about 80 pM, about 1.5 pM to about 70 pM, about 1.5 pM to about 60 pM, about 1.5 pM to about 50 pM, about 1.5 pM to about 40 pM, about 1.5 pM to about 30 pM, about 1.5 pM to about 25 pM, about 1.5 pM to about 20 pM, about 1.5 pM to about 15 pM, about 1.5 pM to about 10 pM, about 1.5 pM to about 5 pM, about 1.5 pM to about 2.5 pM, or about 1.5 pM to about 2 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 0.05 pM, about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM,Page 52 of 10712963423v 1Attorney Docket No.: 2011256-2625 about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM. about 80 pM, about 90 pM. about 100 pM, about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM, or about 250 pM or more. In some embodiments, an additive described herein is added to a medium to a concentration of about 0.05 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 0.1 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 0.25 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 0.5 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 0.75 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1.1 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1.2 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1.3 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1.4 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1.5 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1.6 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1.7 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 1.8 pM. In some embodiments, an additive described herein is added to a medium o a concentration of about 1.9 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 2 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 2.5 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 3 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 3.5 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 4 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 4.5 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 5 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 5.5 pM. In some embodiments, an additive described herein is added to a medium o a concentration of about 6 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 6.5 pM. In somePage 53 of 10712963423v 1Attorney Docket No.: 2011256-2625 embodiments, an additive described herein is added to a medium to a concentration of about 7 |1M. In some embodiments, an additive described herein is added to a medium to a concentration of about 7.5 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 8 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 8.5 |1M. In some embodiments, an additive described herein is added to a medium to a concentration of about 9 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 9.5 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 10 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 11 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 12 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 13 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 14 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 15 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 16 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 17 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 18 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 19 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 20 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 21 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 22 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 23 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 24 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 25 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 30 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 40 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 50 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 60 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 70 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 80 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 90 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 100 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 125 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 150 pM. In some embodiments, anPage 54 of 10712963423v 1Attorney Docket No.: 2011256-2625 additive described herein is added to a medium to a concentration of about 175 p M. In some embodiments, an additive described herein is added to a medium to a concentration of about 200 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 225 pM. In some embodiments, an additive described herein is added to a medium to a concentration of about 250 pM.

[0105] In some embodiments, an additive may be used in combination with one or more additional additives. In some embodiments, an additive may be used in combination with two or more additional additives. In some embodiments, an additive may be used in combination with three or more additional additives. In some embodiments, an additive may be used in combination with four or more additional additives.

[0106] In some embodiments, an additional additive comprises or is any additive provided herein.

[0107] In some embodiments, an additional additive comprises or is a microtubule destabilizing agent.In some embodiments, a microtubule destabilizing agent comprises or is a G2 / M inhibitor. In some embodiments, an additional additive comprises or is a G2 / M inhibitor. In some embodiments, a microtubule destabilizing agent comprises or is colcemid, colchicine, vinblastine, vincristine, or a salt or derivative thereof. In some embodiments, a G2 / M inhibitor comprises or is colcemid, colchicine, vinblastine, vincristine, or a salt or derivative thereof. In some embodiments, an additional additive comprises or is colcemid, colchicine, vinblastine, vincristine, or a salt or derivative thereof.

[0108] In some embodiments, a microtubule destabilizing agent comprises or is colcemid or a salt or derivative thereof. In some embodiments, a G2 / M inhibitor comprises or is colcemid or a salt or derivative thereof. In some embodiments, an additional additive comprises or is colcemid or a salt or derivative thereof. Colcemid may also be referred to as demecolcine, N-methyl-N-deacetyl-colchicine, N-deacetyl- N-methylcolchicine, or (7S)-l,2,3,10-tetramethoxy-7-(methylamino)-6,7-dihydro-5H-benzo[a]heptalen-9- one. Colcemid may have a structure as depicted in the following general formula:

[0109] In some embodiments, colcemid or a salt or derivative thereof is added to a medium to a concentration of about 0.1 pM to about 3.0 pM, about 0.2 pM to about 2.5 pM, about 0.3 pM to about 2.0 pM, about 0.4 pM to about 1.5 pM, about 0.1 pM to about 1.0 pM, about 0.2 pM to about 1.4 pM, about 0.3 pM to about 1.8 pM, about 0.4 pM to about 1.2 pM, or about 0.1 pM to about 1.4 pM. In some embodiments, colcemid or a salt or derivative thereof is added to a medium to a concentration of about 0.1Page 55 of 10712963423v 1Attorney Docket No.: 2011256-2625 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1.0 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2.0 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, or about 3.0 pM.

[0110] In some embodiments, a microtubule destabilizing agent comprises or is colchicine or a salt or derivative thereof. In some embodiments, a G2 / M inhibitor comprises or is colchicine or a salt or derivative thereof. In some embodiments, an additional additive comprises or is colchicine or a salt or derivative thereof. Colchicine may also be referred to as N-[(7S)-l,2,3,10-tetramethoxy-9-oxo-6,7-dihydro-5H- benzo[a]heptalen-7-yl]acetamide. Colchicine may have a structure as depicted in the following general formula:

[0111] In some embodiments, colchicine or a salt or derivative thereof is added to a medium to a concentration of about 1 pM to about 25 pM, about 2 pM to about 20 pM, about 3 pM to about 15 pM, about 4 pM to about 10 pM, or about 5 pM to about 15 pM, about 1 pM to about 8 pM, about 2 pM to about 14 pM, about 3 pM to about 12 pM, about 4 pM to about 22 pM, or about 5 pM to about 25 pM. In some embodiments, colchicine or a salt or derivative thereof is added to a medium to a concentration of about 1.0 pM, about 1.5 pM, about 2.0 pM, about 2.5 pM, about 3.0 pM, about 3.5 pM, about 4.0 pM, about 4.5 pM, about 5.0 pM, about 5.5 pM, about 6.0 pM, about 6.5 pM, about 7.0 pM, about 7.5 pM, about 8.0 pM, about 8.5 pM, about 9.0 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, or about 25 pM.

[0112] In some embodiments, a microtubule destabilizing agent comprises or is vinblastine or a salt or derivative thereof. In some embodiments, a G2 / M inhibitor comprises or is vinblastine or a salt or derivative thereof. In some embodiments, an additional additive comprises or is vinblastine or a salt or derivative thereof. Vinblastine may have a structure as depicted in the following general formula:Page 56 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0113] In some embodiments, vinblastine or a salt or derivative thereof is added to a medium to a concentration of about 0.2 pM to about 10 pM, about 0.4 pM to about 2.0 pM, about 0.6 pM to about 4.0 pM, about 0.8 pM to about 6.0 pM, about 1.0 pM to about 8.0 pM, about 0.4 pM to about 7.0 pM, about 0.6 pM to about 6.0 pM, about 0.8 pM to about 5.0 pM, or about 1.0 pM to about 3.0 pM. In some embodiments, vinblastine or a salt or derivative thereof is added to a medium to a concentration of about0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1.0 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2.0 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3.0 pM, about 3.5 pM, about 4.0 pM, about 4.5 pM, about 5.0 pM, about 5.5 pM, about 6.0 pM, about 6.5 pM, about 7.0 pM, about 7.5 pM, about 8.0 pM, about 8.5 pM, about 9.0 pM, about 9.5 pM, or about 10 pM.

[0114] In some embodiments, a microtubule destabilizing agent comprises or is vincristine or a salt or derivative thereof. In some embodiments, a G2 / M inhibitor comprises or is vincristine or a salt or derivative thereof. In some embodiments, an additional additive comprises or is vincristine or a salt or derivative thereof. Vincristine may also be referred to as leurocristine. Vincristine may have a structure as depicted in the following general formula:

[0115] In some embodiments, vincristine or a salt or derivative thereof is added to a medium to a concentration of about 0.2 pM to about 10 pM, about 0.4 pM to about 2.0 pM, about 0.6 pM to about 4.0Page 57 of 10712963423v 1Attorney Docket No.: 2011256-2625 pM, about 0.8 pM to about 6.0 pM, about 1.0 pM to about 8.0 pM, about 0.4 pM to about 7.0 pM, about 0.6 pM to about 6.0 pM, about 0.8 pM to about 5.0 pM, or about 1.0 pM to about 3.0 pM. In some embodiments, vincristine or a salt or derivative thereof is added to a medium to a concentration of about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1.0 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2.0 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM. about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3.0 pM, about 3.5 pM, about 4.0 pM, about 4.5 pM, about 5.0 pM, about 5.5 pM, about 6.0 pM, about 6.5 pM, about 7.0 pM, about 7.5 pM, about 8.0 pM, about 8.5 pM, about 9.0 pM, about 9.5 pM, or about 10 pM.

[0116] In some embodiments, an additional additive comprises or is a microtubule destabilizing agent described in WO 2022 / 159702, which is herein incorporated by reference in its entirety. In some embodiments, a microtubule destabilizing agent is added to a medium to a concentration described in WO 2022 / 159702.

[0117] In some embodiments, an additional additive comprises or is an antioxidant. In some embodiments, an additional additive comprises or is ferulic acid or a salt or derivative thereof. In some embodiments, an antioxidant comprises or is ferulic acid or a salt or derivative thereof.

[0118] In some embodiments, an additional additive described herein is added to a medium to a concentration of about 0.1 pM to about 250 pM, about 0.1 pM to about 225 pM, about 0.1 pM to about 200 pM, about 0.1 pM to about 175 pM, about 0.1 pM to about 150 pM, about 0.1 pM to about 125 pM, about 0.1 pM to about 100 pM, about 0.1 pM to about 90 pM, about 0.1 pM to about 80 pM, about 0.1 pM to about 70 pM, about 0.1 pM to about 60 pM, about 0.1 pM to about 50 pM, about 0.1 pM to about 40 pM, about 0.1 pM to about 30 pM, about 0.1 pM to about 25 pM, about 0.1 pM to about 20 pM, about 0.1 pM to about 15 pM, about 0.1 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 2.5 pM, about 0.1 pM to about 1 pM, about 0.25 pM to about 250 pM, about 0.25 pM to about 225 pM, about 0.25 pM to about 200 pM, about 0.25 pM to about 175 pM, about 0.25 pM to about 150 pM, about 0.25 pM to about 125 pM, about 0.25 pM to about 100 pM, about 0.25 pM to about 90 pM, about 0.25 pM to about 80 pM, about 0.25 pM to about 70 pM, about 0.25 pM to about 60 pM, about 0.25 pM to about 50 pM, about 0.25 pM to about 40 pM, about 0.25 pM to about 30 pM, about 0.25 pM to about 25 pM, about 0.25 pM to about 20 pM, about 0.25 pM to about 15 pM, about 0.25 pM to about 10 pM, about 0.25 pM to about 5 pM, about 0.25 pM to about 2.5 pM, about 0.5 pM to about 250 pM, about 0.5 pM to about 225 pM, about 0.5 pM to about 200 pM, about 0.5 pM to about 175 pM, about 0.5 pM to about 150 pM, about 0.5 pM to about 125 pM, about 0.5 pM to about 100 pM, about 0.5 pM to about 90 pM, about 0.5 pM to about 80 pM, about 0.5 pM to about 70 pM, about 0.5 pM to about 60 pM, about 0.5 pM toPage 58 of 10712963423v 1Attorney Docket No.: 2011256-2625 about 50 pM, about 0.5 pM to about 40 pM, about 0.5 pM to about 30 pM, about 0.5 pM to about 25 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 2.5 pM, about 1 pM to about 250 pM, about 1 pM to about 225 pM, about 1 pM to about 200 pM, about 1 pM to about 175 pM, about 1 pM to about 150 pM, about 1 pM to about 125 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM. about 1 pM to about 30 pM, about 1 pM to about 25 pM. about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 2.5 pM, about 1 pM to about 2 pM, about 1.5 pM to about 250 pM, about 1.5 pM to about 225 pM, about 1.5 pM to about 200 pM, about 1.5 pM to about 175 pM, about 1.5 pM to about 150 pM, about 1.5 pM to about 125 pM, about 1.5 pM to about 100 pM, about 1.5 pM to about 90 pM, about 1.5 pM to about 80 pM, about 1.5 pM to about 70 pM, about 1.5 pM to about 60 pM, about 1.5 pM to about 50 pM, about 1.5 pM to about 40 pM, about 1.5 pM to about 30 pM, about 1.5 pM to about 25 pM, about 1.5 pM to about 20 pM, about 1.5 pM to about 15 pM, about 1.5 pM to about 10 pM, about 1.5 pM to about 5 pM, about 1.5 pM to about 2.5 pM, about or about 1.5 pM to about 2 pM. In some embodiments, an additional additive described herein is added to a medium to a concentration of about 0.05 pM, about 0.1 pM, about 0.25 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM, or about 250 pM or more.

[0119] In some embodiments, an additive is added to a medium prior to addition of host cells to the medium. In some embodiments, an additive is added to a medium substantially simultaneously to addition of host cells to the medium. In some embodiments, an additive is added to a medium after addition of host cells to the medium.

[0120] In some embodiments, an additive is added to a medium prior to transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium about 1 minute to about 60 minutes, about 15 minutes to about 60 minutes, about 30 minutes to about 60 minutes, about 45 minutes to about 60 minutes, about 1 minute to about 45 minutes, about 15 minutes to about 45 minutes, about 30 minutes to about 45 minutes, about 1 minute to about 30 minutes, about 15 minutes to about 30 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, or 1 minute to about 5 minutesPage 59 of 10712963423v 1Attorney Docket No.: 2011256-2625 prior to transfection with one or more vectors. In some embodiments, an additive is added to a medium about 1 hour to about 48 hours, about 6 hours to about 48 hours, about 12 hours to about 48 hours, about 18 hours to about 48 hours, about 24 hours to about 48 hours, about 30 hours to about 48 hours, about 36 hours to about 48 hours, about 42 hours to about 48 hours, about 1 hour to about 42 hours, about 6 hours to about 42 hours, about 12 hours to about 42 hours, about 18 hours to about 42 hours, about 24 hours to about 42 hours, about 30 hours to about 42 hours, about 36 hours to about 42 hours, about 1 hour to about 36 hours, about 6 hours to about 36 hours, about 12 hours to about 36 hours, about 18 hours to about 36 hours, about 24 hours to about 36 hours, about 30 hours to about 36 hours, about 1 hour to about 30 hours, about 6 hours to about 30 hours, about 12 hours to about 30 hours, about 18 hours to about 30 hours, about 24 hours to about 30 hours, about 1 hour to about 24 hour's, about 6 hours to about 24 hours, about 12 hours to about 24 hours, about 18 hours to about 24 hours, about 1 hour to about 18 hours, about 6 hours to about 18 hours, about 12 hours to about 18 hours, about 1 hour to about 12 hours, about 6 hours to about 12 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, or about 1 hour to about 2 hours prior to transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium about 1 to about 48 hours prior to transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes prior to transfection with one or more vectors. In some embodiments, an additive is added to a medium about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or 48 hours prior to transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes prior to transfection with one or more vectors. In some embodiments, an additive is added to a medium at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or 48 hours prior to transfection of host cells with one or more vectors.

[0121] In some embodiments, an additive is added to a medium substantially simultaneously to transfection of host cells with one or more vectors.Page 60 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0122] In some embodiments, an additive is added to a medium after transfection of host cells with one or more vectors. In some embodiments, an additive is added to a medium about 1 minute to about 60 minutes, about 15 minutes to about 60 minutes, about 30 minutes to about 60 minutes, about 45 minutes to about 60 minutes, about 1 minute to about 45 minutes, about 15 minutes to about 45 minutes, about 30 minutes to about 45 minutes, about 1 minute to about 30 minutes, about 15 minutes to about 30 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, or 1 minute to about 5 minutes after transfection with one or more vectors. In some embodiments, an additive is added to a medium about1 hour to about 24 hours, about 2 hours to about 24 hours, about 4 hours to about 24 hours, about 8 hours to about 24 hours, about 12 hours to about 24 hours, about 16 hours to about 24 hours, about 20 hours to about 24 hours, about 1 hour to about 20 hour's, about 2 hours to about 20 hours, about 4 hours to about 20 hours, about 8 hours to about 20 hours, about 12 hours to about 20 hours, about 16 hours to about 20 hours, about 1 hour to about 16 hours, about 2 hours to about 16 hours, about 4 hours to about 16 hours, about 8 hours to about 16 hours, about 12 hours to about 16 hours, about 1 hour to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 8 hours to about 12 hours, about 1 hour to about 8 hours, about 2 hours to about 8 hours, about 4 hours to about 8 hours, about 1 hour to about 4 hours, about2 hours to about 4 hours or about 1 hour to about 2 hours after transfection with one or more vectors. In some embodiments, an additive is added to a medium about 1 to about 60 minutes after transfection with one or more vectors. In some embodiments, an additive is added to a medium about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes after transfection with one or more vectors. In some embodiments, an additive is added to a medium about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours after transfection with one or more vectors. In some embodiments, an additive is added to a medium at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes after transfection with one or more vectors. In some embodiments, an additive is added to a medium at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours after transfection with one or more vectors.

[0123] In some embodiments, an additive can provide an increased titer (c.g., genome titer) of recombinant viral vector particles (e.g., rAAV particles) produced when added to a medium as comparedPage 61 of 10712963423v 1Attorney Docket No.: 2011256-2625 to a reference condition, e.g., recombinant viral vector particles (e.g., rAAV particles) produced using a medium without the additive. In some embodiments, an increased titer comprises or is an about 1-fold to about 3-fold, about 1-fold to about 2-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 2-fold, or about 2-fold to about 2.5-fold increased titer. In some embodiments, an increased titer comprises or is an about 1-fold to about 3-fold increased titer. In some embodiments, an increased titer comprises or is an about 1-fold or more, 1.25-fold or more, 1.5-fold or more, 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, or 3-fold or more increased titer. In some embodiments, an increased titer comprises or is an about at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, or at least 3-fold increased titer. In some embodiments, an increased titer comprises about 1.0 x 1011vg / ml, about 1.5 x 1011vg / ml, about 2.0 x 1011vg / ml, about 2.5 x 1011vg / ml, about 3.0 x 1011vg / ml, about 3.5 x 1011vg / ml, about 4.0 x 1011vg / ml, about 4.5 x 1011vg / ml, about 5.0 x 1011vg / ml, about 5.5 x 1011vg / ml, about 6.0 x 1011vg / ml, about 6.5 x 1011vg / ml, about 7.0 x 1011vg / ml, about 7.5 x 1011vg / ml, about 8.0 x I011vg / ml, about 8.5 x 1011vg / ml, about 9.0 x 1011vg / ml, about 9.5 x 1011vg / ml, about 1.0 x 1012vg / ml or more.Host Cells

[0124] Among other things, the present disclosure provides host cells for producing recombinant viral vectors (e.g., rAAVs) and / or recombinant viral vector particles (e.g., rAAV particles). Various host cells for production of recombinant viral vectors (e.g., rAAVs) and / or recombinant viral vector particles (e.g., rAAV particles) have been described in Merten. Cell Gene Ther Insights. 2016; 2(5), 521-551, which is hereby incorporated by reference in its entirety. A host cell includes a progeny cell of an original cell transfected with at least one vector described herein. A progeny cell of a parental cell may not be substantially identical in morphology or genomic content as a parent cell due to natural, accidental, and / or deliberate mutation.

[0125] In some embodiments, a host cell comprises or is a kidney cell (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, baby hamster kidney (BHK), or a variant thereof). In some embodiments, a host cell comprises or is a HEK293 cell or a valiant thereof. HEK293 cells and valiants thereof have reportedly been implemented in the production of various recombinant proteins, recombinant viral vectors (e.g., rAAVs), and recombinant viral vector particles (e.g., rAAV particles). See, e.g., Tan et al. Front Bioeng Biotechnol. 2021 ;9:796991 , which is hereby incorporated by reference in its entirety. In some embodiments, a host cell comprises or is a HEK293 cell or a variant thereof. In some embodiments, a host cell comprises or is HEK293T, HEK293F, HEK293FT, HEK293FTM, HEK293SG, HEK293H, HEK293E, HEK293A, or a variant thereof. See, e.g., Yuan ct al. Biomcd Pharmacol J. 2018;l 1(2), which is hereby incorporated by reference in its entirety. In some embodiments, a HEK293 cell or a valiant thereofPage 62 of 10712963423v 1Attorney Docket No.: 2011256-2625 comprises or expresses an El polypeptide. In some embodiments, a host cell comprises or is a BHK cell or a variant thereof.

[0126] In some embodiments, a host cell comprises or is a Chinese hamster ovary (CHO) cell (e.g., CHO KI, DXB-11 CHO, Veggie-CHO, or a variant thereof). CHO cells and variants thereof have reportedly been implemented in the production of various recombinant proteins, including particularly antibodies, e.g., monoclonal antibodies. See, e.g., Tihanyi and Nyitray. Drug Discov Today Technol. 2020;38:25-34. Recent reports have also demonstrated usage of CHO cells in the production of recombinant viral vectors (e.g., rAAVs) and recombinant viral vector particles (e.g., rAAV particles). See, e.g., Cao et al. Biotechnol Bioeng. 2024;121(l):395-402. and Nagy et al. Sci Rep. 2023;13(l):19210., which are hereby incorporated by reference in their entirety.

[0127] In some embodiments, a host cell comprises or is a HeLa cell or a variant thereof. In some embodiments, a host cell comprises or is a HeLaS3 cell or a variant thereof. Various methods and systems for production of recombinant viral vectors (e.g., rAAVs) and recombinant viral vector particles (e.g., rAAV particles) have been reported. See, e.g., Escandell et al. Biotechnol Bioeng. 2023;120(9):2578-2587. and Merten. Cell Gene Therapy Insights 2016; 2(5), 521-551., which are hereby incorporated by reference in their entirety.

[0128] In some embodiments, a host cell comprises or is an insect cell (e.g., Sf9, Sf21, or a variant thereof). In some embodiments, a host cell comprises or is a Sf9 cell or a variant thereof. In some embodiments, a host cell comprises or is a baculovirus-infected insect cell, e.g., a baculovirus-infected Sf9 cell. Various Sf9 cell lines suitable for production of recombinant viral vectors (e.g., rAAVs) and recombinant viral vector particles (e.g., rAAV particles) have been reported. See, e.g., Mietzsch et al. Hum Gene Ther Methods. 2017;28(l): 15-22., which is hereby incorporated by reference in its entirety.

[0129] In some embodiments, a host cell comprises or is a COS cell (e.g., COS-7 or a variant thereof). In some embodiments, a host cell comprises or is a retinal cell. In some embodiments, a host cell comprises or is a Vero cell. In some embodiments, a host cell comprises or is a CV1 cell. In some embodiments, a host cell comprises or is a HepG2 cell. In some embodiments, a host cell comprises or is a WI38 cell. In some embodiments, a host cell comprises or is a MRC5 cell. In some embodiments, a host cell comprises or is a Colo205 cell. In some embodiments, a host cell comprises or is a HB 8065 cell. In some embodiments, a host cell comprises or is a HL-60 cell. In some embodiments, a host cell comprises or is a Jurkat cell. In some embodiments, a host cell comprises or is a Daudi cell. In some embodiments, a host cell comprises or is an A431 epidermal cell. In some embodiments, a host cell comprises or is a CV-1 cell. In some embodiments, a host cell comprises or is a U937 cell. In some embodiments, a host cell comprises or is a 3T3 cell. In some embodiments, a host cell comprises or is an L cell. In some embodiments, a host cell comprises or is a C127 cell. In some embodiments, a host cell comprises or is a SP2 / 0 cell. In somePage 63 of 10712963423v 1Attorney Docket No.: 2011256-2625 embodiments, a host cell comprises or is a NS-0 cell. In some embodiments, a host cell comprises or is a MMT060562 cell. In some embodiments, a host cell comprises or is a Sertoli cell. In some embodiments, a host cell comprises or is a BRL3A cell. In some embodiments, a host cell comprises or is a HT1080 cell. In some embodiments, a host cell comprises or is a myeloma cell. In some embodiments, a host cell comprises or is a tumor cell. In some embodiments, a host cell comprises or is a cell line or variant derived from any cell described herein.

[0130] In some embodiments, a host cell comprises or is a packaging cell. In some embodiments, a packaging cell is a packaging cell for production of rAAV. A packaging cell for production of rAAV reportedly comprises, e.g., stably comprises, a nucleic acid sequence encoding at least one Rep polypeptide and a nucleic acid sequence encoding at least one Cap polypeptide. In some embodiments, a packaging cell for production of rAAV comprises a nucleic acid sequence encoding at least one Rep polypeptide and a nucleic acid sequence encoding at least one Cap polypeptide. A packaging cell may be of any packaging cell line available. Various packaging cell lines, including those for production of rAAV, are known in the art. See, e.g., Merten. Microorganisms. 2024;12(2):384., which is hereby incorporated by reference in its entirety. In some embodiments, a packaging cell line is a cell line comprising any cell type described herein. In some embodiments, a packaging cell line is a HeLa cell line or a variant thereof. In some embodiments, a packaging cell line is an A549 cell line or a variant thereof. In some embodiments, a packaging cell line is a HEK293 cell line or a variant thereof. In some embodiments, a packaging cell line is a Sf9 cell line or a variant thereof. In some embodiments, a packaging cell is a cell of any cell type described herein. In some embodiments, a packaging cell is a HeLa cell or a variant thereof. In some embodiments, a packaging cell is an A549 cell or a variant thereof. In some embodiments, a packaging cell is a HEK293 cell or a variant thereof. In some embodiments, a packaging cell is a Sf9 cell or a variant thereof.

[0131] In some embodiments, a host cell comprises or is a producer cell. In some embodiments, a producer cell is a producer cell for production of rAAV. A producer cell reportedly comprises, e.g., stably comprises, a nucleic acid sequence encoding at least one Rep polypeptide, a nucleic acid sequence encoding at least one Cap polypeptide, and a nucleic acid sequence encoding at least one payload flanked by an ITR on either side of the at least one payload. In some embodiments, a producer cell comprises, e.g., stably comprises, a nucleic acid sequence encoding at least one Rep polypeptide, a nucleic acid sequence encoding at least one Cap polypeptide, and a nucleic acid sequence encoding at least one payload flanked by an ITR on either side of the at least one payload. A producer cell may be of any producer cell line available. Various producer cell lines, including those for production of rAAV, are known in the art. See, e.g., Merten. Microorganisms. 2024;12(2):384., which is hereby incorporated by reference in its entirety. In some embodiments, a producer cell line is a HeLa cell line or a variant thereof. In some embodiments, a producerPage 64 of 10712963423v 1Attorney Docket No.: 2011256-2625 cell line is a cell line comprising any cell type described herein. In some embodiments, a producer cell line is an A549 cell line or a variant thereof. In some embodiments, a producer cell line is a HEK293 cell line or a variant thereof. In some embodiments, a producer cell line is a Sf9 cell line or a variant thereof. In some embodiments, a producer cell is a cell of any cell type described herein. In some embodiments, a producer cell is a HeLa cell or a variant thereof. In some embodiments, a producer cell is an A549 cell or a valiant thereof. In some embodiments, a producer cell is a HEK293 cell or a valiant thereof. In some embodiments, a producer cell is a Sf9 cell or a variant thereof.

[0132] Expression of at least one Rep polypeptide, at least one Cap polypeptide, and / or at least one helper polypeptide in a packaging or a producer cell may be able to be induced. A producer cell may comprise one or more inducible genetic elements that control the expression of a nucleic acid sequence encoding at least one Rep polypeptide and / or a nucleic acid sequence encoding at least one helper polypeptide. In some embodiments, a producer cell comprises one or more inducible genetic elements operably linked to a nucleic acid sequence encoding at least one Rep polypeptide. In some embodiments, a producer cell comprises one or more inducible genetic elements operably linked to a nucleic acid sequence encoding at least one Cap polypeptide. In some embodiments, a producer cell comprises one or more inducible genetic elements operably linked to a nucleic acid sequence encoding at least one helper polypeptide. In some embodiments, a producer cell comprises one or more inducible genetic elements operably linked to a nucleic acid sequence encoding at least one Rep polypeptide, at least one Cap polypeptide, and / or a nucleic acid sequence encoding at least one helper polypeptide.

[0133] Induction of expression of at least one Rep polypeptide, at least one Cap polypeptide, and / or at least one helper polypeptide in a packaging cell or a producer cell may occur through one of a number of mechanisms as reported in the ai t. For example, in some embodiments, induction of expression of at least one Rep polypeptide and / or at least one Cap polypeptide in a packaging cell is through expression of at least one helper polypeptide, in some embodiments, induction of expression of at least one Rep polypeptide and / or at least one Cap polypeptide in a packaging cell is through transfection of one or more vectors encoding at least one helper polypeptide. In some embodiments, induction of expression of at least one Rep polypeptide and / or at least one Cap polypeptide in a packaging cell is through infection with one or more viruses, e.g., an adenovirus-AAV hybrid virus and / or an adenoviral helper virus. In some embodiments, induction of expression of at least one Rep polypeptide and / or at least one Cap polypeptide in a producer cell is through expression of at least one helper polypeptide. In some embodiments, induction of expression of at least one Rep polypeptide and / or at least one Cap polypeptide in a producer cell is through infection with one or more viruses, e.g., a helper virus, e.g., an adenoviral helper virus. In some embodiments, induction of expression of at least one Rep polypeptide and / or at least one Cap polypeptide in a producer cell is through a helper virus-free induction system, e.g., a Cre / Lox-based system or induciblePage 65 of 10712963423v 1Attorney Docket No.: 2011256-2625 promoter system. Various induction methods for inducing expression of at least one Rep polypeptide, at least one Cap polypeptide, and / or at least one helper polypeptide in a packaging and / or producer cell are reviewed in Merten. Microorganisms. 2024;12(2):384., which is hereby incorporated by reference in its entirety.

[0134] In some embodiments, a host cell comprises or is an adherent cell. In some embodiments, host cells comprise or are adherent cells. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 99%, or more host cells in culture are adherent cells.

[0135] In some embodiments, a host cell comprises or is a suspension cell. In some embodiments, host cells comprise or are suspension cells. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 99%, or more host cells in culture are suspension cells.Vectors

[0136] Among other things, the present disclosure provides vectors for producing recombinant viral vector particles, e.g., rAAV particles. Many forms of vectors can be used in methods, compositions, and systems described herein for producing recombinant viral vector particles, e.g., rAAV particles. Nonlimiting examples of vectors for producing recombinant viral vector particles, e.g., rAAV particles, include, but are not limited to, plasmids, bacteriophage vectors, cosmids, phagemids, and artificial chromosomes.

[0137] In some embodiments, a vector encodes at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload (e.g., for expression of an inhibitory nucleic acid or polypeptide described herein). In some embodiments, a vector encodes at least one Rep polypeptide. In some embodiments, a vector encodes least one Cap polypeptide. In some embodiments, a vector encodes at least one helper polypeptide.

[0138] In some embodiments, a vector encodes at least one Rep polypeptide and at least one Cap polypeptide. In some embodiments, a vector encodes at least one Cap polypeptide and at least one pay load flanked by an ITR on either side of the at least one payload. In some embodiments, a vector encodes at least one helper polypeptide and at least one Rep polypeptide. In some embodiments, a vector encodes at least one helper polypeptide and at least one payload flanked by an ITR on cither side of the at least one payload. In some embodiments, a vector encodes at least one Rep polypeptide and at least one payloadPage 66 of 10712963423v 1Attorney Docket No.: 2011256-2625 flanked by an ITR on either side of the at least one payload. In some embodiments, a vector encodes at least one helper polypeptide and at least one Cap polypeptide.

[0139] In some embodiments, methods described herein comprise transfecting a host cell with at least three vectors. In some embodiments, three vectors comprise: (i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload, (ii) a second vector encoding at least one Rep polypeptide and at least one Cap polypeptide, and (iii) a third vector encoding at least one helper polypeptide. In some embodiments, methods described herein comprises transfecting a host cell with at least two vectors. In some embodiments, at least two vectors comprise: (i) a first vector encoding at least one Cap polypeptide and at least one payload flanked by an ITR on either side of the at least one payload; and (ii) a second vector encoding at least one helper polypeptide and at least one Rep polypeptide.

[0140] A vector can include conventional control elements operably linked to a nucleic acid encoding any payload or polypeptide described herein, in a manner that permits transcription, translation and / or expression in a cell transfected with a vector. Expression control sequences include, but are not limited to, transcription, initiation, termination, promoter, and / or enhancer sequences; efficient RNA processing signals, such as splicing and / or polyadenylation (polyA) signals (e.g., a rabbit [3-globin polyA signal); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., a Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of an encoded product. In some embodiments, a vector includes other regulatory elements, such as at least one Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE).

[0141] A number of expression control sequences, including promoters that are native, constitutive, inducible, and / or tissue-specific, are known in the art and may be included in a vector described herein. Examples of constitutive promoters include, but are not limited to, a retroviral Rous sarcoma virus (RSV) LTR promoter (e.g., further comprising an RSV enhancer), a cytomegalovirus (CMV) promoter (e.g., further comprising an CMV enhancer), an SV40 promoter, and a dihydrofolate reductase promoter.

[0142] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors, such as temperature, or the presence of a specific physiological state (e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only). Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include a zinc-inducible sheep metallothionine (MT) promoter, a dexamethasone (Dex)- inducible mouse mammary tumor virus (MMTV) promoter, a T7 polymerase promoter system, an ecdysone insect promoter, a tetracycline-repressible system, a tetracycline-inducible system, a RU486-induciblc system, and an rapamycin-inducible system. Still other types of inducible promoters that may be useful arePage 67 of 10712963423v 1Attorney Docket No.: 2011256-2625 regulated by a specific physiological state, such as temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0143] In some embodiments, a native promoter or fragment thereof for a nucleic acid encoding any pay load or polypeptide described herein may be used. Other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression.Vectors encoding at least one Payload

[0144] The present disclosure, among other things, provides vectors (e.g., plasmids) encoding at least one payload. A payload sequence is generally a sequence of interest to be introduced into a cell, tissue, organ, or organism. An rAAV vector comprises cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses,” ed., P. Tijsser, CRC Press, pp. 155-168 (1990), which is hereby incorporated by reference in its entirety).

[0145] As used herein, the terms “5”’ and “3”’ are relative terms to define a spatial relationship or directionality between two or more segment of a nucleic acid sequence. Thus, 3’ of a nucleic acid indicates a segment of the nucleic acid that is downstream of another segment, while 5’ indicates a segment of the nucleic acid that is upstream of another segment. For example, 3’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 3’ end of the nucleic acid sequence. Similarly, 5’ may indicate that a segment is in the 5’ half of the nucleic acid sequence or even at the 5’ end of the nucleic acid sequence. Unless indicated otherwise, the directionality of a nucleic acid will be in the 5’ to 3’ direction of translation.

[0146] ITR sequences are typically about 115 to 145 nt in length. In some embodiments, one or both of a 5’ ITR or a 3’ ITR nucleic acid sequence are modified via insertion, deletion and / or substitution relative to a known ITR nucleic acid sequence, e.g., the 145 nt wild-type 5’ and 3’ ITRs from an AAV2 genome. Modification of ITR nucleic acid sequences is known to those of skill in the art (See, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996), each of which is hereby incorporated by reference in its entirety). AAV ITR sequences may be obtained from any known AAV, including mammalian AAV types, e.g., an AAV2 genome.

[0147] In some embodiments, a payload comprises or is a heterologous nucleic acid with a therapeutic purpose, e.g., a miRNA, siRNA, shRNA, mRNA, snRNA, CRISPR / Cas guide RNA (gRNA), or a precursor thereof. In some embodiments, a payload comprises or is a heterologous protein with a therapeutic purpose, e.g., an enzyme, cytokine, antibody, receptor, fusion protein, or chimeric polypeptide.Page 68 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0148] In some embodiments, a pay load is linked to a secretion signal sequence for secretion of an expressed polypeptide. One of skill in the art will recognize that a payload can be selected from any heterologous polypeptide or nucleic acid of interest. In some embodiments, a payload sequence comprises one or more aptamer-binding domains or polypeptide-binding domains (e.g., transcription factor-binding domains). A vector will also typically include other regulatory elements (e.g., promoters, introns, and / or enhancers) to regulate expression or amount of a payload in a cell or tissue.

[0149] In some embodiments, a payload sequence can be of any length, e.g., between about 2 nucleotides and about 10,000 nucleotides in length or any integer value there between. In some embodiments, a nucleic acid sequence encoding a payload comprises at least 20 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides. at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1600 nucleotides, at least 1700 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 8000 nucleotides, or at least 9000 nucleotides. In some embodiments, a nucleic acid sequence encoding a payload comprises between about 50 and 500 nucleotides in length, between about 100 and 1,000 nucleotides in length, between about 250 and 2,000 nucleotides in length, between about 150 and 3,000 nucleotides in length, between about 500 and 6,000 nucleotides in length, between about 700 and 7,000 nucleotides in length, between about 1,000 and 8,000 nucleotides in length, or between about 2,000 and 5,000 nucleotides in length.

[0150] An exemplary payload of interest comprises at least one inhibitory nucleic acid (e.g., at least one miRNA, siRNA, shRNA, gRNA, or any combination thereof) that inhibits expression of a protein. In some embodiments, a vector comprises one or more nucleic acids encoding at least one miRNA that inhibits expression of a protein. In some embodiments, a target nucleic acid sequence encoding a protein comprises or is a wild-type nucleic acid sequence, or a mutant or variant thereof. In some embodiments, targeted nucleic acid sequences include or are mRNA sequences. In some embodiments, targeted mRNA sequences comprise or are human mRNA.

[0151] In some embodiments, a payload comprises or is a polypeptide with a therapeutic purpose. In some embodiments, a vector comprises one or more nucleic acids for insertion of a nucleic acid sequence encoding a polypeptide with a therapeutic purpose into a genome of a subject (e.g., a human), e.g., for long-Page 69 of 10712963423 vlAttorney Docket No.: 2011256-2625 term expression of a polypeptide in a subject. In some embodiments, a subject has reduced expression of a polypeptide relative to a subject with a corresponding wild-type gene. In some embodiments, a subject has a mutant polypeptide relative to a subject with a corresponding wild-type gene. In some embodiments, a subject has a dysfunctional polypeptide relative to a subject with a corresponding wild-type gene.Vectors encoding at least one Rep Polypeptide

[0152] The present disclosure, among other things, provides vectors (e.g., plasmids) encoding at least one Rep polypeptide. Rep polypeptides are involved in viral DNA replication, resolution of replicative intermediates, and generation of single-stranded genomes. In some embodiments, a vector comprises a nucleic acid sequence encoding one, two, three, or more (e.g., all) of Rep78, Rep68, Rep52, or Rep40, or a variant thereof.

[0153] In some embodiments, a Rep polypeptide comprises a nucleic acid sequence derived from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV-HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV- PHP.N, or AAV.CAP-B1 to AAV.CAP-B25 serotype, or a variant of any of the foregoing. For example, a nucleic acid sequence encoding a Rep polypeptide may be derived from a known AAV genome sequence including, but not limited to, AAV1 Accession No. NC_002077 or AF063497; AAV2 Accession No. NC_001401; AAV3A Accession No. NC_001729; AAV3B Accession No. NC_001863; AAV4 Accession No. NC_001829; AAV5 Accession No. Y18065 or AF085716; Accession No. AAV6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, or NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; or Bovine AAV NC_005889, AY388617.

[0154] In some embodiments, a nucleic acid sequence encoding a Rep polypeptide is derived from an AAV genome sequence or a variant thereof as described in US Patent Nos. 7,906,111; 6,759,237; 7,105,345; 7,186,552; 9,163,260; 9,567,607; 4,797,368; 5,139,941; 5,252,479; 6,261,834; 7,718,424; 8,507,267; 8,846,389; 6,984,517; 7,479,554; 6,156,303; 8,906,675; 7,198,951 ; 10,041 ,090; 9,790,472; 10,308,958; 10,526,617; 7,282,199; 7,790,449; 8,962,332; 9,587,250;10,590,435; 10,265,417; 10,485,883; 7,588,772; 8,067,01; 8,574,583; 8,906,387; 8,734,809; 9,284,357; 10,035,825; 8,628,966; 8,927,514; 9,623,120; 9,777,291; 9,783,825; 9,803,218; 9,834,789; 9,839,696; 9,585,971; or 10,519,198; U.S. Publication Nos. 2017 / 0166926; 2019 / 0015527; 2019 / 0054188; or 2020 / 0080109; or International Publication Nos. WO2018 / 160582, W02020 / 028751, or W02020 / 068990, each of which is hereby incorporated by reference in its entirety.

[0155] In some embodiments, a promoter is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide. In some embodiments, a wild-type promoter of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV-Page 70 of 10712963423v 1Attorney Docket No.: 2011256-2625HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV-PHP.N, or AAV.CAP-B1 to AAV.CAP-B25, or a variant of any of the foregoing is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding at least one Rep polypeptide comprises a p5 and / or pl9 promoter.Vectors encoding at least one Cap Polypeptide

[0156] The present disclosure, among other things, provides vectors (e.g., plasmids) encoding at least one Cap polypeptide. Cap polypeptides (e.g., VP1, VP2, and VP3) are structural polypeptides that form a capsid. In some embodiments, a vector comprises a nucleic acid sequence encoding one, two, or three of VP1, VP2, and VP3.

[0157] In some embodiments, a Cap polypeptide comprises a nucleic acid sequence derived from an AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV-HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV- PHP.N, or AAV.CAP-B1 to AAV.CAP-B25 serotype, or a variant of any of the foregoing. For example, a nucleic acid sequence encoding a Cap polypeptide may be derived from a known AAV genome sequence including, but not limited to, AAV1 Accession No. NC_002077 or AF063497; AAV2 Accession No. NC_001401; AAV3A Accession No. NC_001729; AAV3B Accession No. NC_001863; AAV4 Accession No. NC_001829; AAV5 Accession No. Y18065 or AF085716; Accession No. AAV6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, or NC_004828; Avian AAV strain DA-1 NC_006263. AY629583; or Bovine AAV NC_005889, AY388617.

[0158] In some embodiments, a nucleic acid sequence encoding a Cap polypeptide is derived from an AAV genome sequence or a variant thereof as described in US Patent Nos. 7,906,111; 6,759,237; 7,105,345; 7,186,552; 9,163,260; 9,567,607; 4,797,368; 5,139,941; 5,252,479; 6,261,834; 7,718,424; 8,507,267; 8,846,389; 6,984,517; 7,479,554; 6,156,303; 8,906,675; 7,198,951; 10,041,090; 9,790,472; 10,308,958; 10,526,617; 7,282,199; 7,790,449; 8,962,332; 9,587,250;10,590,435; 10,265,417; 10,485,883; 7,588,772; 8,067,01; 8,574,583; 8,906,387; 8,734,809; 9,284,357; 10,035,825; 8,628,966; 8,927,514; 9,623,120; 9,777,291; 9,783,825; 9,803,218; 9,834,789; 9,839,696; 9,585,971; or 10,519,198; U.S. Publication Nos. 2017 / 0166926; 2019 / 0015527; 2019 / 0054188; or 2020 / 0080109; or International Publication Nos. WO2018 / 160582, W02020 / 028751, or W02020 / 068990, each of which is hereby incorporated by reference in its entirety.

[0159] In some embodiments, a capsid comprises or is a modified capsid protein (e.g., a capsid comprising a modified VP3 region). Methods of producing modified capsid proteins are known in the art (Sec, e.g., US20130310443, which is hereby incorporated by reference in its entirety). In some embodiments, a modified capsid protein comprises at least one non-native amino acid substitution at aPage 71 of 10712963423v 1Attorney Docket No.: 2011256-2625 position that corresponds to a surface-exposed amino acid (e.g., a surface exposed tyrosine) in a wild-type capsid protein. In some embodiments, a modified capsid protein comprises a non-tyrosine amino acid (e.g., a phenylalanine) at a position that corresponds to a surface-exposed tyrosine amino acid in a wild-type capsid protein, a non-threonine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed threonine amino acid in a wild-type capsid protein, a non-lysine amino acid (e.g., a glutamic acid) at a position that corresponds to a surface-exposed lysine amino acid in a wild-type capsid protein, a non-serine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed serine amino acid in a wildtype capsid protein, or a combination thereof. In some embodiments, a modified capsid protein comprises or has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0160] In some embodiments, a promoter is operably linked to a nucleic acid sequence encoding at least one Cap polypeptide. In some embodiments, a wild-type promoter of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV- HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV-PHP.N, or AAV.CAP-B1 to AAV.CAP-B25, or a val iant of any of the foregoing is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide. In some embodiments, a p40 promoter is operably linked to a nucleic acid sequence encoding at least one Cap polypeptide.Vectors encoding at least one Helper Polypeptide

[0161] The present disclosure, among other things, provides vectors (e.g., plasmids) encoding at least one helper polypeptide. AAV is a helper-dependent DNA parvovirus, which belongs to the genus Dependovirus. Production of recombination AAV requires co-infection with a related virus (e.g., adenovirus, herpes, or vaccinia virus) or a helper vector encoding helper polypeptides, such as structural proteins and proteins for viral genome replication.

[0162] A vector encoding at least one helper polypeptide can comprise nucleotide sequences for non- AAV derived viral and / or cellular functions upon which AAV is dependent for replication, which may include, but are not limited to, activation of gene transcription, stage-specific mRNA splicing, DNA replication, synthesis of at least one Cap polypeptide, and / or capsid assembly. Viral-based helper polypeptides can be derived from any known helper virus, such as adenovirus, herpesvirus, vaccinia virus, or a combination thereof. Thus, a vector (e.g., a plasmid) encoding at least one helper polypeptide can comprise sufficient helper function to permit packaging of an rAAV vector into capsid polypeptides. In some embodiments, a helper polypeptide comprises an Ad5 helper polypeptide. In some embodiments, a nucleic acid sequence of an Ad5 helper polypeptide is derived from Adenovirus 5 genome (GenBank Accession No. AY601635). In some embodiments, a helper polypeptide comprises an Ad2 helper polypeptide.Page 72 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0163] Helper polypeptides (e.g., Ad5 or Ad2 helper polypeptides) can comprise at least one, two, three, or more (e.g., all) of El, E2A, E4orf6, or VA RNA. In some embodiments, El comprises Ela and / or Elb. In some embodiments, one or both of E2A and VA RNA increase stability and / or efficiency of AAV mRNA translation, such as for cap gene transcripts. In some embodiments, E4orf6 facilitates DNA replication. In some embodiments, Ela comprises a transactivator (e.g., regulating activity of at least one Ad gene, AAV rep gene, and / or AAV cap gene). In some embodiments, Elb comprises a viral mRNA transport. Helper polypeptides are described in further detail in Coura and Nardi, Genetics and Molecular Biology, 31(1): 1-11 (2008), which is hereby incorporated by reference in its entirety.Transfection

[0164] Among other things, the present disclosure provides methods for transfection of host cells with one or more vectors described herein for producing recombinant viral vector particles, e.g., rAAV particles. Host cells (e.g., mammalian cells, such as HEK293 cells, CHO cells, HeLa cells, or variants thereof) can be transfected with one or more vectors encoding: (i) at least one payload (e.g., an inhibitory nucleic acid or a polypeptide) flanked by an inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one Rep polypeptide, (iii) at least one Cap polypeptide, and (vii) at least one helper polypeptide. In some embodiments, transfection comprises or is transient transfection. In some embodiments, transfection comprises or is stable transfection. In some embodiments, host cells are transfected with two or three vectors described herein.

[0165] Transfection can include any method known to a skilled person for introducing one or more exogenous nucleic acids into host cells (e.g., mammalian cells, such as HEK293 cells, CHO cells, HeLa cells, or valiants thereof). For example, transfection can include, but is not limited to, vector-based transfection, plasmid-based transfection, virus-based transfection, electroporation, lipofection (e.g., with one or more cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, cationic polymer-based transfection (e.g., using DEAE-dextran and / or polyethylenimine (PEI)), or a combination of any of the foregoing. Various transfection reagents are commercially available to those skilled in the art, including, e.g., FectoVIR (e.g., FectoVIR-AAV, FectoVIR-LV) transfection reagent (Polyplus), Lipofectamine (e.g., Lipofectamine, Lipofectamine 2000, Lipofectamine 3000) transfection reagent (Invitrogen), PEIpro transfection reagent (Polyplus), TransIT-VirusGEN transfection reagent (MirusBio). Various transfection techniques are generally known in those skilled in the art. See, e.g., Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981) Gene 13:197, each of which is hereby incorporated by reference in its entirety.Page 73 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0166] In some embodiments, host cells (e.g., mammalian cells, such as HEK293 cells, CHO cells, HeLa cells, or variants thereof) are transfected with a transfection reagent. In some embodiments, a transfection reagent comprises or is a polymer. In some embodiments, a transfection reagent comprises or is a cationic polymer. In some embodiments, a transfection reagent comprises or is PEI, calcium phosphate, a lipid capable of traversing a cell membrane (e.g., a liposome or a micelle), a nanoparticle, or a combination thereof. In some embodiments, a transfection reagent comprises or is PEI. In some embodiments, a transfection reagent comprises or is FectoVIR (e.g., FectoVIR-AAV, FectoVIR-LV) transfection reagent. In some embodiments, a transfection reagent comprises or is Lipofectamine (e.g., Lipofectamine, Lipofectamine 2000, Lipofectamine 3000) transfection reagent. In some embodiments, a transfection reagent comprises or is PEIpro transfection reagent. In some embodiments, a transfection reagent comprises or is TransIT-VirusGEN transfection reagent.Culturing

[0167] Among other things, the present disclosure provides methods for culturing host cells described herein for producing of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles.

[0168] Cultures of host cells can be prepared in any medium suitable for a particular host cell type being cultured. Exemplary components of host cell medium can include, but are not limited to, inorganic salts, carbohydrates (e.g., sugars, such as glucose, galactose, maltose, fructose), amino acids, vitamins (e.g., B group vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, biotin), fatty acids (e.g., cholesterol, steroids, various fatty acids, e.g., linoleic acid), proteins (e.g., albumin, transferrin, fibronectin, fetuin), serum (e.g., albumins, growth factors, or growth inhibitors, such as fetal bovine serum, newborn calf serum, or horse serum), trace elements (e.g., zinc, copper, selenium, tricarboxylic acid intermediates), hydrolysates (e.g., derived from plant or animal sources), or combinations thereof.

[0169] Commercially available media can be used for culturing host cells described herein. Exemplary media can include, but are not limited to, Dulbecco's Modified Eagle’s Medium (DMEM, Sigma), FreeStyle™ F17 Expression Medium (ThermoFisher), DMEM / F12 medium (Invitrogen), CD OptiCHO™ medium (Invitrogen), CD EfficientFeed™ media (Invitrogen), Cell Boost (HyClone™) media (GE Life Sciences), BalanCD™ CHO Feed (Irvine Scientific), BD Recharge™ (Becton Dickinson), Cellvento Feed™ (EMD Millipore), Ex-cell CHOZN Feed™ (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma- Aldrich), SheffCHO™ (Kerry), Zap-CHO™ (Invitria), ActiCHO™ (PAA / GE Healthcare), Minimal Essential Medium (Sigma), or RPML1640 (Sigma).

[0170] A medium, e.g., a commercially available medium, can be supplemented as necessary with, e.g., hormones and / or other growth factors (e.g., insulin, transferrin, epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine,Page 74 of 10712963423v 1Attorney Docket No.: 2011256-2625 thymidine), antibiotics (e.g., kanamycin, puromycin, neomycin, hygromycin, blasticidin, gentamycin, Grl8, zeocin), trace elements (e.g., zinc, copper, selenium, tricarboxylic acid intermediates), lipids (e.g., cholesterol, steroids, various fatty acids, e.g., linoleic acid), or carbohydrates (e.g., sugars, such as glucose, galactose, maltose, fructose). These necessary supplements can be added at appropriate concentrations as known to those skilled in the art. In some embodiments, a medium for culturing host cells described herein comprises glutamine or a glutamine dipeptide. In some embodiments, a medium for culturing host cells described herein comprises a surfactant. In some embodiments, a medium comprises or is a serum-free medium, a protein-free medium, or a chemically defined medium. Any other necessary supplements can also be included at appropriate concentrations that as known to those skilled in the art.

[0171] In some embodiments, the present disclosure provides methods for producing a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles. In some embodiments, the present disclosure provides a method for producing a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors, (b) adding an additive to the medium, and (c) culturing the host cells under conditions suitable for production of the plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles. In some embodiments, the present disclosure provides a method for producing a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors encoding (i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one Rep polypeptide, (iii) at least one Cap polypeptide, and (iv) at least one helper polypeptide; (b) adding an additive to the medium; and (c) culturing the host cells under conditions suitable for production of the plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles. In some embodiments, a method for producing a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles further comprises collecting the produced plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles from the culture.

[0172] In some embodiments, the present disclosure provides a method for producing a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors, (b) adding an additive to the medium, (c) culturing the host cells under conditions suitable for production of the plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles, and (d) collecting the produced plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles from the culture. In some embodiments, the present disclosure provides a method for producing a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particlesPage 75 of 10712963423v 1Attorney Docket No.: 2011256-2625 comprising (a) transfecting one or more host cells in a culture comprising a medium with one or more vectors encoding (i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one Rep polypeptide, (iii) at least one Cap polypeptide, and (iv) at least one helper polypeptide; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of the plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles; and (d) collecting the produced plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles from the culture.

[0173] In some embodiments, the present disclosure provides a method for producing a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least on Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; and (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles. In some embodiments, the present disclosure provides a method for producing a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least on Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles; and (d) collecting the produced plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles from the culture. In some embodiments, the present disclosure provides a method for producing a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least on Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; and (c) culturing the host cells under conditions suitable for production of the plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles; wherein the one or more host cells each independently comprise (i) a nucleic acid sequence encoding at least one payload flanked by an ITR on either side of the at least one payload; (ii) a nucleic acid sequence encoding at least one Rep polypeptide; and (iii) a nucleic acid sequence encoding at least one Cap polypeptide. In some embodiments, the present disclosure provides a method for producing a plurality of rAAV particles comprising (a) inducing expression of at least one Rep polypeptide and at least on Cap polypeptide in one or more host cells in a culture comprising a medium; (b) adding an additive to the medium; (c) culturing the host cells under conditions suitable for production of the plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles; and (d) collecting the produced plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles from the culture; wherein the one or more host cells each independently comprise (i) a nucleic acid sequence encoding at least one payload flanked by an ITR on cither side of the at least one payload; (ii) a nucleic acid sequence encoding at least one Rep polypeptide; and (iii) a nucleic acid sequence encoding at least onePage 76 of 10712963423v 1Attorney Docket No.: 2011256-2625Cap polypeptide.

[0174] Collection of a produced plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles from a culture can involve various methods, e.g. recovery methods known in the ait. In some embodiments, viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles are collected from culture supernatant. In some embodiments, viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles are collected by lysing host cells and recovering the viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles from lysate, e.g., after centrifugation.

[0175] In some embodiments, lysis of host cells uses a lysis solution. Various lysis solutions are known in the art. In some embodiments, a lysis solution comprises a detergent, e.g., sodium dodecyl sulfate (SDS), ethyl trimethyl ammonium bromide, Triton X-100, bile salts (e.g., cholate), zwitterionic detergents (e.g., CHAPS). In some embodiments, a lysis solution comprises one or more salts (e.g., NaCl). In some embodiments, a lysis solution has a pH of greater than about 7.

[0176] In some embodiments, viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles are purified. Various purification methods are known in the art. In some embodiments, viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles are purified using centrifugation, e.g., ultracentrifugation. In some embodiments, viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles are purified using filtration, e.g., UF / DF filtration. In some embodiments, viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles are purified using chromatography, e.g., liquid chromatography, high-performance liquid chromatography, affinity chromatography, ion-exchange chromatography (e.g., cation exchange chromatography or anion exchange chromatograph). In some embodiments, chromatography, e.g., affinity chromatography, uses a virus-specific binding resin, e.g., an AAV-specific binding resin. Various AAV-specific binding resins are commercially available, including, e.g., AVB Sepharose High Performance resin (GE Healthcare), POROS CaptureSelect resins (Thermo Fisher).

[0177] Host cells described herein can be cultured in a cell culture vessel or bioreactor. In some embodiments, host cells are cultured in a cell culture vessel. Examples of cell culture vessels include dishes, plates, and flasks. In some embodiments, a cell culture vessel is used for culturing adherent cells. In some embodiments, a cell culture vessel is used for culturing suspension cells. Exemplary cell culture vessels include, but are not limited to, 35mm, 60mm, 100mm, or 150mm dishes; multi-well plates (e.g., 6- well plates, 12-well plates, 24-well plates, 48-well plates, 96 well plates); flasks (e.g., T-flasks, e.g., T-25 flasks, T-75 flasks, T-160 flasks); or shaker flasks.

[0178] In some embodiments, host cells described herein arc cultured in a biorcactor. In some embodiments, a bioreactor is used for culturing adherent cells. In some embodiments, a bioreactor is usedPage 77 of 10712963423v 1Attorney Docket No.: 2011256-2625 for culturing suspension cells. In some embodiments, a bioreactor comprises or is a continuous flow batch bioreactor. In some embodiments, a bioreactor comprises or is a perfusion bioreactor. In some embodiments, a bioreactor comprises or is a batch process bioreactor. In some embodiments, a bioreactor comprises or is a fed batch bioreactor. A bioreactor can be maintained under conditions sufficient to produce viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles. Culture conditions in a bioreactor can be modulated to optimize yield, purity, and / or structure of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles.

[0179] In some embodiments, a plurality of re viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles are produced in a large-scale preparation comprising host cells, e.g., host cells described herein. In some embodiments, a large-scale preparation of a plurality of viral vector particles, e.g., recombinant viral vector particles, e.g., rAAV particles is produced in a bioreactor. In some embodiments, a bioreactor comprises at least about 1 liter of culture media, e.g., between about 1 liter to about 2,000 liters, about 1,000 liters to about 2,000 liters, about 500 liters to about 1,500 liters, about 250 liters to about 1,250 liters, about 1,500 liters to about 2,000 liters, about 1,000 liters to about 1,500 liters, about 500 liters to about 1,750 liters, about 250 liters to about 1,500 liters, about 1,500 liters to about 2,000 liters, about 10 liters to about 800 liters, about 25 liters to about 500 liters, about 2 liters to about 300 liters, about 100 liters to about 400 liters, about 50 liters to about 600 liters, about 15 liters to about 800 liters, about 80 liters to about 500 liters, about 20 liters to about 300 liters, about 100 liters to about 400 liters, or about 50 liters to about 600 liters of culture media, e.g., at least about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 10 liters, about 15 liters, about 20 liters, about 25 liters, about 30 liters, about 35 liters, about 40 liters, about 45 liters, about 50 liters, about 55 liters, about 60 liters, about 65 liters, about 70 liters, about 75 liters, about 80 liters, about 85 liters, about 90 liters, about 100 liters, about 125 liters, about 150 liters, about 175 liters, about 200 liters, about 225 liters, about 250 liters, about 275 liters, about 300 liters, about 350 liters, about 400 liters, about 450 liters, about 500 liters, about 550 liters, about 575 liters, 600 liters, about 625 liters, about 650 liters, about 675 liters, about 700 liters, about 725 liters, about 750 liters, about 775 liters, about 800 liters, about 850 liters, about 900 liters, about 950 liters, or about 1,000 liters of culture media.

[0180] In some embodiments, a bioreactor is maintained under conditions that promote growth of a host cell described herein, e.g., at a temperature (e.g., about 37° C) and gas concentration (e.g., 5% to 10% carbon dioxide) that is permissive for host cell growth. For example, a bioreactor can perform one or more of the following: feeding of nutrients and / or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and carbon dioxide levels, maintenance of pH level, agitation (e.g., stirring), cleaning, and / or sterilization.Page 78 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0181] Exemplary bioreactor units may contain multiple reactors within a unit, e.g., a unit can comprise 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more bioreactors. Any suitable bioreactor diameter and / or shape can be used. In some embodiments, suitable reactors can be round, e.g., cylindrical. In some embodiments, suitable reactors can be square, e.g., rectangular'.Viral Vectors

[0182] Among other things, the present disclosure provides methods, compositions, and systems for producing viral vector particles, e.g., recombinant viral vector particles, e.g., recombinant adeno-associated virus (rAAV) particles. Recombinant viral vectors and particles thereof have many uses including, e.g., introducing various payloads (e.g., exogenous genes) into target cells, e.g., mammalian cells, e.g., human cells.

[0183] Various viral vectors, e.g., recombinant viral vectors, have been described in the art. Nonlimiting examples of viral vectors, e.g., recombinant viral vectors, include adenovirus, adeno-associated virus (AAV), alphavirus, herpesvirus, retrovirus, and vaccina virus.

[0184] In some embodiments, a recombinant viral vector comprises or is an adenoviral vector. An adenovirus may comprise or be from any origin, serotype, subgroup, subtype, or combination thereof. For example, an adenovirus, e.g., a human adenovirus can be of subgroup A (e.g., serotypes 12, 18, 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, 50, 55), subgroup C (e.g., 1, 2, 5, 6, 57), subgroup D (e.g., serotypes 8, 9. 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26. 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43. 44, 45, 46, 47, 48, 49, 51, 53. 54, 56, 58, 59, 60, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73 74, 75). subgroup E (e.g., serotype 4), subgroup F (e.g., serotype 40, 41), or subgroup G (serotype 52). In some embodiments, an adenoviral vector comprises or is a human adenovirus. In some embodiments, an adenoviral vector comprises or is a mammalian adenovirus. In some embodiments, an adenoviral vector comprises or is a simian adenovirus. In some embodiments, an adenoviral vector is a replication-deficient adenoviral vector. In some embodiments, an adenoviral vector is a helper dependent adenoviral (HDAd) vector. In some embodiments, an adenoviral vector is a chimeric adenoviral vector. A chimeric adenoviral vector may comprise portions, e.g., capsid proteins, from two or more subgroups and / or serotypes. Various adenoviral vectors, e.g., recombinant adenoviral vectors that may be used in gene therapy are described in the art. See, e.g., Wold and Toth. Curr Gene Ther. 2013;13(6):421-433, which is hereby incorporated by reference in its entirety.

[0185] In some embodiments, a recombinant viral vector comprises or is an adeno-associated viral (AAV) vector. AAV systems are generally well known in the art; for example, see, Kelleher and Vos, Biotcchniqucs, 17(6): 1110-17 (1994); Cotten ct al., P.N.A.S. U.S.A., 89(13) :6094-98 (1992); Curiel, Nat Immun, 13(2-3):141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and AsokanPage 79 of 10712963423v 1Attorney Docket No.: 2011256-2625A, et al., Mol. Ther., 20(4):699-708 (2012), each of which is hereby incorporated by reference in its entirety. Methods for generating and using AAV vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is hereby incorporated by reference in its entirety.

[0186] Generally, AAV vectors for use in methods, compositions, and systems described herein may be of any AAV serotype. AAV serotypes generally have different tropisms to infect different tissues. In some embodiments, an AAV serotype is selected based on a tropism. Several AAV serotypes have been characterized including, but not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV-HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV-PHP.N, or AAV.CAP-B1 to AAV.CAP-B25, as well as variants or hybrids thereof. For example, in some embodiments, an AAV vector comprises or is an AAV2 / 5, AAV2 / 6, AAV2 / 8 or AAV2 / 9 vector (e.g., AAV6, AAV8 or AAV9 serotype having AAV2 ITR). In some embodiments, an AAV serotype may have or comprise a mutation in an AAV9 sequence (e.g., as described in N Pulicherla et al. Molecular Therapy 19(6): 1070-1078 (2011), which is hereby incorporated by reference in its entirety). AAV9 serotypes may include, but not limited to, AAV9.68, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, and AAV9.84. In some embodiments, an AAV9 variant comprises or is AAVhu68 or a variant thereof (e.g., as described in WO 2018 / 160585, which is hereby incorporated by reference in its entirety). Other AAV vectors are described in, e.g., Sharma et al., Brain Res Bull. 2010 Feb 15; 81(2-3): 273, which is hereby incorporated by reference in its entirety.

[0187] An AAV vector may comprise or be based on a serotype selected from any following serotypes or variants thereof including, but not limited to, AAV9.68, AAV1, AAV10, AAV106.1 / hu.37, AAV11, AAV114.3 / hu.4O, AAV 12, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.1 / hu.43, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu.l l, AAV16.3, AAV16.8 / hu.l0, AAV161.10 / hu.60, AAV161.6 / hu.61, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2, AAV2.5T, AAV2- 15 / rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2- 3 / rh.61, AAV24.1 , AAV2-4 / rh.5O, AAV2-5 / rh.51 , AAV27.3, AAV29.3 / bb. 1, AAV29.5 / bb.2, AAV2G9, AAV-2- pre-miRNA-101, AAV3, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-l l / rh.53, AAV3-3, AAV33.12 / hu.l7, AAV33.4 / hu.l5, AAV33.8 / hu.l6, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4, AAV4-19 / rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-lb, AAV42-2, AAV42-3a, AAV42- 3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42- 6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV4-8 / rl 1.64, AAV4-8 / rh.64, AAV4-9 / rh.54, AAV5, AAV52.1 / hu.2O, AAV52 / hu.l9, AAV5- 22 / rh.58, AAV5-3 / rh.57, AAV54.1 / hu.21, AAV54.2 / hu.22, AAV54.4R / hu.27, AAV54.5 / hu.23, AAV54.7 / hu.24, AAV58.2 / hu.25, AAV6, AAV6.1, AAV6.1.2, AAV6.2, AAV7, AAV7.2, AAV7.3 / hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11, AAV9.13,Page 80 of 10712963423v 1Attorney Docket No.: 2011256-2625AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.84, AAV9.9, AAVA3.3, AAVA3.4, AAV A3.5, AAV A3.7, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl. AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV- DJ, AAV-DJ8, AAVF3, AAVF5, AAV-h, AAVH-l / hu.l, AAVH2, AAVH-5 / hu.3, AAVH6, AAVhEl.l, AAVhER1.14, AAVhErl.16, AAVhErl.18, AAVhER1.23, AAVhErl.35, AAVhErl.36, AAVhErl.5, AAVhErl.7, AAVhErl.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1. AAVhu.E AAVhu.10, AAVhu.l E AAVhu.12, AAVhu.13, AAVhu.14 / 9. AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.2, AAVhu.20. AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52. AAVhu.53. AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57. AAVhu.58. AAVhu.6, AAVhu.6O, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.tl9, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVLG-9 / hu.39, AAV- LKO1, AAV-LK02, AAVLK03, AAV-LKO3, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV- LKO8, AAV-LKO9, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV- LK17, AAV-LK18, AAV-LK19, AAVN721-8 / rh.43, AAV-PAEC, AAV-PAEC11, AAV- PAEC12, AAV- PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC 8. AAVpi.l, AAVpi.2, AAVpi.3. AAVrh.lO, AAVrh.12, AAVrh.13. AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.5O, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.6O, AAVrh.6E AAVrh.62, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, BAAV, B P61 AAV, B P62 AAV, B P63 AAV, bovine AAV, caprine AAV, Japanese AAV10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK 16, AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and AAV SM 10-8.

[0188] In some embodiments, an AAV may be or comprise a serotype generated from an AAV9 capsid library with mutations in amino acids 390 to 627 (VP1 numbering), c.g., as described in Pulichcrla ct al. (Molecular' Therapy 19(6): 1070-1078 (2011), which is hereby incorporated by reference in its entirety. AnPage 81 of 10712963423v 1Attorney Docket No.: 2011256-2625AAV serotype (with corresponding nucleotide and amino acid substitutions) may include, but is not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A I 3I 4T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G 1203 A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T5821), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54 (CI 531 A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L51 II), AAV9.65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80 (G1441A,;G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87 (T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L), and AAV9.95 (T1605A; F535L).

[0189] An AAV serotype may be from any number of species. For example, an AAV may be or comprise an avian AAV (AAAV), e.g., as described in U.S. Patent No. 9,238,800, which is hereby incorporated by reference in its entirety. An AAV serotype may be or comprise a bovine AAV (BAAV), e.g., as described in U.S. Patent Nos. 9,193,769 or 7,427,396, each of which is hereby incorporated by reference in its entirety. An AAV may be or comprise a caprine AAV, e.g., as described in U.S. Patent No. 7427396, which is hereby incorporated by reference in its entirety. An AAV serotype may also be a variant or hybrid of any of the foregoing.

[0190] In some embodiments, an AAV vector is derived from an AAV genome sequence or a variant thereof as described in U.S. Patent Nos. 7,906,111; 6,759,237; 7,105,345; 7,186,552; 9,163,260; 9,567,607; 4,797,368; 5,139,941 ; 5,252,479; 6,261,834; 7,718,424; 8,507,267; 8,846,389; 6,984,517; 7,479,554; 6,156,303; 8,906,675; 7,198,951; 10,041,090; 9,790,472; 10,308,958; 10,526,617; 7,282,199; 7,790,449; 8,962,332; 9,587,250;10,590,435; 10,265,417; 10,485,883; 7,588,772; 8,067,01; 8,574,583; 8,906,387; 8,734,809; 9,284,357; 10,035,825; 8,628,966; 8,927,514; 9,623,120; 9,777,291; 9,783,825; 9,803,218;Page 82 of 10712963423v 1Attorney Docket No.: 2011256-26259,834,789; 9,839,696; 9,585,971; or 10,519,198; U.S. Publication Nos. 2017 / 0166926; 2019 / 0015527; 2019 / 0054188; or 2020 / 0080109; or International Publication Nos. WO 2018 / 160582, WO 2020 / 028751, or WO 2020 / 068990, each of which is hereby incorporated by reference in its entirety.

[0191] In some embodiments, an AAV vector comprises or is a naturally occurring AAV. In some embodiments, an AAV vector is a modified AAV or a variant of a naturally occurring AAV. In some embodiments, an AAV vector may be generated by directed evolution, e.g., by DNA shuffling, peptide insertion, or random mutagenesis, in order to introduce modifications into the AAV sequence to improve one or more properties for gene therapy. In some embodiments, such modifications avoid or lessen an immune response or recognition by neutralizing antibodies and / or allow for more efficient and / or targeted transduction (see, e.g., Asuri et al., Molecular Therapy 20.2 (2012): 329-338, which is hereby incorporated by reference in its entirety). Methods of using directed evolution to engineer an AAV vector can be found, e.g., in U.S. Patent No.: 8,632,764, which is hereby incorporated by reference in its entirety. In some embodiments, a modified AAV is modified to include a specific tropism.

[0192] In some embodiments, an AAV vector may be a dual or triple AAV vector, e.g., for the delivery of large payloads (e.g., payloads of greater than approximately 5kb) and / or to address safety concerns associated with administration of single AAV vectors. In some embodiments, a dual AAV vector may include two separate AAV vectors, each including a fragment of a full sequence of a large pay load of interest, and when recombined, the fragments form the full sequence of the large payload of interest or a functional portion thereof. In some embodiments, a triple AAV vector may include three separate AAV vectors, each including a fragment of a sequence of a large payload of interest, and when recombined, the fragments form the full sequence of the large payload of interest or a functional portion thereof.

[0193] Multiple AAV vectors (e.g., dual or triple AAV vectors) can be delivered to and co-transduced into the same cell, where fragments of a payload of interest recombine and generate a single mRNA transcript of the entire payload of interest. In some embodiments, fragmented payloads include nonoverlapping sequences. In some embodiments, fragmented payloads include specified overlapping sequences. In some embodiments, multiple AAV vectors for dual or triple transfection may be the same type of AAV vector (e.g., same serotype and / or same construct). In some embodiments, multiple AAV vectors for dual or triple transfection may be different types of AAV vector (e.g., different serotype or different construct).

[0194] In some embodiments, an AAV vector comprises a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector. In some embodiments, an AAV vector comprises an expression construct and one or more regions comprising ITR sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking an expression construct. In some embodiments, an AAV vector is cncapsidatcd by a viral capsid. In some embodiments, a viral capsid comprises 60 capsid protein subunits. In somePage 83 of 10712963423v 1Attorney Docket No.: 2011256-2625 embodiments, a viral capsid comprises VP1, VP2, and VP3. In some embodiments, VP1, VP2, and VP3 subunits are present in a capsid at a ratio of about 1:1:10, respectively.

[0195] In some embodiments, an AAV vector comprises a capsid including modified capsid proteins (e.g., capsid proteins comprising a modified VP3 region). Methods of producing modified capsid proteins are known in the art (see, e.g., US20130310443, which is hereby incorporated by reference in its entirety). In some embodiments, an AAV vector comprises a modified capsid protein comprising at least one nonnative amino acid substitution at a position that corresponds to a surface-exposed amino acid (e.g., a surface exposed tyrosine) in a wild-type capsid protein. In some embodiments, an AAV vector comprises a modified capsid protein comprising a non-tyrosine amino acid (e.g., a phenylalanine) at a position that corresponds to a surface-exposed tyrosine amino acid in a wild-type capsid protein, a non-threonine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed threonine amino acid in a wild-type capsid protein, a non-lysine amino acid (e.g., a glutamic acid) at a position that corresponds to a surface- exposed lysine amino acid in a wild-type capsid protein, a non-serine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed serine amino acid in a wild-type capsid protein, or a combination thereof. In some embodiments, an AAV vector comprises a capsid that includes modified capsid proteins having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0196] ITR sequences of an AAV vector can be derived from any AAV serotype (e.g., AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV-HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV-PHP.N, or AAV.CAP- B1 to AAV.CAP-B25, or variants or hybrids thereof). In some embodiments, ITR sequences are derived from one or more other serotypes, e.g., as described in US Patent Nos. 7,906,111; 6,759,237; 7,105,345; 7,186,552; 9,163,260; 9,567,607; 4,797,368; 5,139,941; 5,252,479; 6,261,834; 7,718,424; 8,507,267; 8,846,389; 6,984,517; 7,479,554; 6,156,303; 8,906,675; 7,198,951; 10,041,090; 9,790,472; 10,308,958; 10,526,617; 7,282,199; 7,790,449; 8,962,332; 9,587,250;10,590,435; 10,265,417; 10,485,883; 7,588,772; 8,067,01 ; 8,574,583; 8,906,387; 8,734,809; 9,284,357; 10,035,825; 8,628,966; 8,927,514; 9,623,120; 9,777,291; 9,783,825; 9,803,218; 9,834,789; 9,839,696; 9,585,971; or 10,519,198; U.S. Publication Nos. 2017 / 0166926; 2019 / 0015527; 2019 / 0054188; or 2020 / 0080109; or International Publication Nos. WO 2018 / 160582, WO 2020 / 028751, or WO 2020 / 068990, each of which is hereby incorporated by reference in its entirety. ITR sequences and plasmids containing ITR sequences are known in the art and are commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; and described in Kessler et al. PNAS. 1996 Nov 26;93(24): 14082-7; and U.S. Pat. Nos. 5,139,941 and 5,962,313; each of which is hereby incorporated by reference in its entirety).

[0197] Additional methods for generating and isolating AAV viral vectors suitable for various uses,Page 84 of 10712963423v 1Attorney Docket No.: 2011256-2625 e.g., administration or delivery to a subject, arc described in. e.g., U.S. Patent No. 7,790,449; U.S. Patent No. 7,282,199; WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; and U.S. Patent No. 7,588,772, each of which are hereby incorporated by reference in their entirety.

[0198] In some embodiments, a recombinant viral vector comprises or is an alphaviral vector. Alphaviruses are reportedly single-stranded RNA viruses that belong to the Togaviridae family. Nonlimiting examples of alphaviruses include Aura virus, Barmah Forest virus, Bebaru virus, Caaingua virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Eilat virus, Everglades virus, Fort Morgan virus, Getah virus. Highlands J virus, Madariaga virus, Mayaro virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O’nyong’nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Sindbis virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus. In some embodiments, an alphaviral vector is a replicationproficient alphaviral vector. In some embodiments, an alphaviral vector is a replication-deficient alphaviral vector. Research has been conducted on utilization of Ross River virus, Semliki Forest virus, Sindbis virus, and Venezuelan equine encephalitis virus as recombinant viral vectors. See, e.g., Lundstrom. Viruses. 2015;7(5):2321-2333., which is hereby incorporated by reference in its entirety.

[0199] In some embodiments, a recombinant viral vector comprises or is a herpesviral vector. Nonlimiting examples of herpesviruses include cytomegalovirus (CMV), Epstein-Barr virus, and herpes simplex virus (e.g., herpes simplex virus-1 (HSV-1)). CMV is reportedly a double-stranded DNA virus that has been investigated for usage as a viral vector for vaccines or gene therapy. See, e.g., Zeng et al. Viruses. 2023;15(10):2043., which is hereby incorporated by reference in its entirety. In some embodiments, a CMV vector is a murine CMV (MCMV) vector. In some embodiments, a CMV vector is a rhesus CMV (RhCMV) vector. In some embodiments, a CMV vector is a human CMV (HCMV) vector.

[0200] In some embodiments, a recombinant viral vector comprises or is a retroviral vector. Retroviruses are enveloped viruses that belong to viral family Retroviridae. Protocols for production of replication-deficient retroviruses are known in the art; for example, see Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991), each of which is hereby incorporated by reference in its entirety. A number of retroviral systems are known in the art; for example, see U.S. Patent Nos. 5,994,136, 6,165,782, and 6,428,953, each of which is hereby incorporated by reference in its entirety. In some embodiments, a retrovirus comprises or is a lentivirus of Retroviridae family. In some embodiments, a lentivirus comprises or is human immunodeficiency viruses (e.g., HIV-1 or HIV-2), simian immunodeficiency virus (S1V), feline immunodeficiency virus (FIV), equine infections anemia (EIA), or visna virus.Page 85 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0201] In some embodiments, a recombinant viral vector comprises or is a vaccina viral vector. Vaccina virus is reportedly an enveloped virus with a linear, double-stranded DNA genome of approximately 190kb in length. In some embodiments, a vaccina viral vector is a replication-proficient vaccina viral vector. In some embodiments, a vaccina viral vector is a replication-deficient vaccina viral vector. Development of vaccina virus as a recombinant viral vector has included studies regarding usage as an oncolytic virus for treatment of various cancers and / or tumors and usage as a viral vaccine vector. See, e.g., Zhang et al. Hum Vaccin Immunother. 2021 ; 17(6): 1578-1585., which is hereby incorporated by reference in its entirety.Compositions

[0202] Among other things, the present disclosure provides a composition comprising a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by methods described herein. In some embodiments, a composition is a liquid composition. In some embodiments, a composition is a pharmaceutical composition.

[0203] In some embodiments, a composition comprises a pharmaceutical composition comprising at least one pharmaceutically acceptable component (e.g., a pharmaceutically acceptable carrier, diluent, or excipient). Such pharmaceutical compositions are useful for, among other things, administration, e.g., to a cell or a subject, e.g., in vivo or ex vivo.

[0204] In some embodiments, pharmaceutical compositions also contain a pharmaceutically acceptable carrier, excipient, or diluent. Such excipients include any pharmaceutical agent, e.g., a pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids, such as water, saline, glycerol, sugars, and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, or sulfates; and the salts of organic acids, such as acetates, propionates, malonates, or benzoates. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such vehicles.

[0205] Pharmaceutical compositions may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, or succinic. Salts tend to be more soluble in aqueous or other protonic solvents than corresponding free base forms. In some embodiments, a pharmaceutical composition may be a lyophilized powder.

[0206] Pharmaceutical compositions can include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, and isotonic and absorption promoting or delaying agents,Page 86 of 10712963423v 1Attorney Docket No.: 2011256-2625 compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules, and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral, and antifungal agents) can also be incorporated into the compositions.

[0207] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0208] Compositions suitable for parenteral administration can comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal, vegetable, or synthetic oils. Aqueous injection suspensions may contain substances that increase the viscosity of a suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, suspension may also contain suitable stabilizers or agents that increase solubility to allow for preparation of highly concentrated solutions.

[0209] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and poly viny IpyiTolidone .

[0210] After pharmaceutical compositions have been prepared, they may be placed in an appropriate container and labeled for treatment. Such labeling can include amount, frequency, and method of administration.

[0211] Various pharmaceutical compositions and delivery systems appropriate for the compositions, methods and uses of the disclosure are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy. 21st Edition. Philadelphia, PA. Lippincott Williams & Wilkins, 2005).Uses

[0212] Among other things, the present disclosure provides methods of administering or delivering a viral vector, e.g., a recombinant viral vector, e.g., a rAAV produced by a method provided herein. In somePage 87 of 10712963423v 1Attorney Docket No.: 2011256-2625 embodiments, a method comprises administering or delivering a recombinant viral vector, e.g., a rAAV, produced by a method provided herein to a cell, a tissue, or an organism described herein. In some embodiments, a method of administering or delivering a recombinant viral vector, e.g., a rAAV, produced by a method provided herein comprises administering or delivering the recombinant viral vector, e.g., the rAAV, to a cell, a tissue, or an organism. In some embodiments, a method comprises administering or delivering a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by a method provided herein to a cell, a tissue, or an organism described herein. In some embodiments, a method of administering or delivering a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by a method provided herein comprises administering or delivering the plurality of recombinant viral vector particles, e.g., rAAV particles, to a cell, a tissue, or an organism. In some embodiments, a method comprises administering or delivering a composition, e.g., a pharmaceutical composition, comprising a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by a method provided herein to a cell, a tissue, or an organism described herein. In some embodiments, a method of administering or delivering a composition, e.g., a pharmaceutical composition, comprising a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by a method provided herein comprises administering or delivering the composition, e.g., the pharmaceutical composition, to a cell, a tissue, or an organism.

[0213] In some embodiments, a method of gene therapy comprises administration or delivery of a recombinant viral vector, e.g., a rAAV, produced by a method provided herein. In some embodiments, a method of gene therapy comprises administration or delivery of a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by a method provided herein. In some embodiments, a method of gene therapy comprises administration or delivery of a composition, e.g., a pharmaceutical composition, comprising a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by a method provided herein. In some embodiments, a method of gene therapy comprises administering or delivering a recombinant viral vector, e.g., a rAAV, produced by a method provided herein to a subject. In some embodiments, a method of gene therapy comprises administering or delivering a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by a method provided herein to a subject. In some embodiments, a method of gene therapy comprises administering or delivering a composition, e.g., a pharmaceutical composition, comprising a plurality of recombinant viral vector particles, e.g., rAAV particles, produced by a method provided herein to a subject.

[0214] In some embodiments, a cell is a eukaryotic cell. In some embodiments, a cell is a mammalian cell. In some embodiments, a cell is a human cell. In some embodiments, a cell is a cell collected from a subject. In some embodiments, a cell is a cell collected from a human subject.

[0215] In some embodiments, a tissue is a eukaryotic tissue. In some embodiments, a tissue is a mammalian tissue. In some embodiments, a tissue is a human tissue. In some embodiments, a tissue is aPage 88 of 10712963423v 1Attorney Docket No.: 2011256-2625 tissue collected from a subject. In some embodiments, a tissue is a tissue collected from a human subject.

[0216] In some embodiments, an organism is an animal. In some embodiments, an organism is a mammal. In some embodiments, an organism is a mouse. In some embodiments, an organism is a rat. In some embodiments, an organism is a non-human primate. In some embodiments, an organism is a human.

[0217] In some embodiments, a subject is an animal. In some embodiments, a subject is a mammal. In some embodiments, a subject is a mouse. In some embodiments, a subject is a rat. In some embodiments, a subject is a non-human primate. In some embodiments, a subject is a human. In some embodiments, a subject is an adult. In some embodiments, a subject is a human adult. In some embodiments, a subject is a child. In some embodiments, a subject is a human child. In some embodiments, a subject is suffering from or susceptible to a condition, disorder, or disease. In some embodiments, a subject is suffering from a condition, disorder, or disease. In some embodiments, a subject is susceptible to a condition, disorder, or disease.

[0218] In some embodiments, a condition, disorder, or disease comprises or is a cardiac condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a dermatological condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is an endocrine condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a gastric condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a hepatic condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is an immune condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is an inflammatory condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a metabolic condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a muscular condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a neoplastic condition, disorder, or disease, e.g., a cancer or a tumor. In some embodiments, a condition, disorder, or disease comprises or is a neurological condition, disorder, or disease, e.g., a neurodegenerative condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is an ocular condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a respiratory condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a skeletal condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a urinary condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a vascular condition, disorder, or disease.

[0219] In some embodiments, a condition, disorder, or disease comprises or is a hereditary condition, disorder, or disease. In some embodiments, a condition, disorder, or disease comprises or is a sporadic condition, disorder, or disease.Page 89 of 10712963423v 1Attorney Docket No.: 2011256-2625EXAMPLES

[0220] Certain examples of provided technologies are presented herein. Those skilled in the art appreciate that many technologies can be utilized to prepare and / or assess properties and / or activities of provided technologies in accordance with the present disclosure.Example 1. Various compounds can modulate titers of produced rAAV particles.

[0221] Various compounds were tested as additives for addition to a medium comprising host cells for production of rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml in 5 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in a 24-deep well plate. Cells were transfected on day 2 with a payload vector, a Rep / Cap vector, and a helper vector. Various compounds (BX-795, parthenolide, ruxolitinib, TPCA-1, momelotinib (CYT387), trichostatin A (TSA)) were added to the cultures at various concentrations (for BX-795, parthenolide, ruxolitinib, TPCA-1, and momelotinib, 0.5, 1, 4, and 10 |1M; for TSA, 0.05, 0.1, 0.4, and 1 pM) at either 30 min post-transfection or 24 hrs post-transfection. Control cultures that did not receive any compound and control cultures that received only DMSO at various concentrations (0.05, 0.1, 0.4, and 1 pM) were also examined. At 3 days post-addition of the various compounds, cells were harvested and lysed and resulting lysates were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates and titer fold increase as compared to control cultures that did not receive any compound was calculated. Certain exemplary results are displayed in Figure 1.

[0222] As shown in Figure 1, various compounds, when used as an additive added to a medium comprising host cells, can provide modulation of titer of produced rAAV as compared to a control condition without an additive added. In some embodiments, addition of momelotinib to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without momelotinib added. In some embodiments, addition of momelotinib to a medium after transfection of host cells in the medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without momelotinib added. In some embodiments, addition of BX-795 to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without BX-795 added. In some embodiments, addition of BX-795 to a medium after transfection of host cells in the medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without BX-795 added.Example 2. BX-795 and momelotinib can modulate titers of produced rAAV particles.

[0223] BX-795 and momelotinib were tested as additives for addition to a medium comprising host cells for production of rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml in 5 ml Freestyle Fl 7 media (Thermo Fisher) supplemented with 4 mMPage 90 of 10712963423v 1Attorney Docket No.: 2011256-2625 glutamine and 2 g / 1 P188 in a 24-deep well plate. Cells were transfected on day 2 at a cell density of approximately 4 x 106viable cells / ml with a payload vector, a Rep / Cap vector, and a helper vector. BX- 795 or momelotinib (CYT387) was added to the cultures at various concentrations (for BX-795, 0.1, 0.2, 0.8, 1.7, and 3.3 pM; for momelotinib, 0.125,0.25, 1, 2.5, and 5 pM) at 30 min post-transfection. Control cultures that did not receive any compound were also examined. At 3 days post-addition of the various compounds, cells were harvested and lysed and resulting lysates were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates and titer fold increase as compared to control cultures that did not receive any compound was calculated. Certain exemplary results are displayed in Figure 2.

[0224] As shown in Figure 2, BX-795 or momelotinib, when used as an additive added to a medium comprising host cells, can provide modulation of titer of produced rAAV as compared to a control condition without an additive added. In some embodiments, addition of momelotinib to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without momelotinib added. In some embodiments, addition of momelotinib to a medium after transfection of host cells in the medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without momelotinib added. In some embodiments, addition of BX-795 to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without BX-795 added. In some embodiments, addition of BX-795 to a medium after transfection of host cells in the medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without BX-795 added.Example 3. Various compounds can modulate titers of produced rAAV particles.

[0225] Various compounds were tested as additives for addition to a medium comprising host cells for production of rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml in 5 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 Pl 88 in a 24-deep well plate. Cells were transfected on day 2 with a payload vector, a Rep / Cap vector, and a helper vector. At 30 min post-transfection, various compounds (BX-795, parthenolide, ruxolitinib, TPCA-1, momelotinib, trichostatin A, interferon- a-IFNa-R interaction inhibitor, tosylate salt, imidazolo- oxindole, CGP 57380, BMS-345541, spermine, NF-KB activation inhibitor, IMD 0354, lactacystin, BAY 11-7082, epoxomicin, MLN-4924) were added to the cultures at various concentrations as shown in Figure 3. Control cultures that did not receive any compound were also examined. At 3 days post-addition of the various compounds, cells were harvested and lysed and resulting lysates were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates and normalized to rAAV genome titer from control cultures that did not receive any compound. Certain exemplary results are displayed in Figure 3.Page 91 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0226] As shown in Figure 3, various compounds, when used as an additive added to a medium comprising host cells, can provide modulation of titer of produced rAAV as compared to a control condition without an additive added. In some embodiments, addition of momelotinib to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without momelotinib added. In some embodiments, addition of BX-795 to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without BX-795 added. In some embodiments, addition of parthenolide to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without parthenolide added. In some embodiments, addition of ruxolitinib to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without ruxolitinib added. In some embodiments, addition of trichostatin A to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without trichostatin A added. In some embodiments, addition of tosylate salt to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without tosylate salt added. In some embodiments, addition of NF-KB activation inhibitor to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without NF-KB activation inhibitor added. In some embodiments, addition of interferon-a-IFNa- R interaction inhibitor to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without interferon-a-IF a-R interaction inhibitor added. In some embodiments, addition of BMS-345541 to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without BMS-345541 added.Example 4. Various compounds can modulate titers of produced rAAV particles.

[0227] Various compounds were tested as additives for addition to a medium comprising host cells for production of rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml in 15 ml Freestyle F17 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 P188 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a payload vector, a Rep / Cap vector, and a helper vector. At 30 min post-transfection, various compounds (ruxolitinib, interferon-a- IFNa-R interaction inhibitor, NF-KB activation inhibitor, BMS-345541) were added to the cultures at various concentrations as shown in Figure 4. Control cultures that did not receive any compound were also examined. At 3 days post-addition of the various compounds, cells were harvested and lysed and resulting lysates were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Certain exemplary results are displayed in Figure 4 and the table below.Page 92 of 10712963423v 1Attorney Docket No.: 2011256-2625

[0228] As shown in Figure 4, various compounds, when used as an additive added to a medium comprising host cells, can provide modulation of titer of produced rAAV as compared to a control condition without an additive added. In some embodiments, addition of ruxolitinib to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without ruxolitinib added. In some embodiments, addition of interferon-a-IFNa-R interaction inhibitor to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without interferon-a-IFNa-R interaction inhibitor added. In some embodiments, addition of BMS-345541 to a medium can provide an increased titer of produced rAAV as compared to a reference condition, e.g., a medium without BMS-345541 added.Example 5. Various compounds can modulate titers of produced rAAV particles.

[0229] Momelotinib, ruxolitinib, and BX-795 were tested as additives for addition to a medium comprising host cells for production of rAAV particles. In brief, suspension HEK293 cells were inoculated on day 0 at a cell density of 1 x 106cells / ml for AAV2 production or 0.75 x 106cells / ml for AAV9 production in 15 ml Freestyle Fl 7 media (Thermo Fisher) supplemented with 4 mM glutamine and 2 g / 1 P188 in an Ambr 15 bioreactor. Cells were transfected on day 2 with a payload vector, a Rep / Cap vector, and a helper vector. At 30 min post-transfection, momelotinib was added to the cultures at various concentrations as shown in Figure 5, Figure 6, and Figure 7. A control culture without momelotinib added was also examined. At 4 days post-transfection, cells were harvested and lysed and resulting lysates from cells for production of AAV2 were supplemented with MgCh, NaCl, and benzonase. rAAV genome titer was determined using ddPCR of the resultant lysates. Certain exemplary results are displayed in Figure 5, Figure 6, Figure 7, and the table below.

[0230] As shown in Figure 5, momelotinib, when used as an additive added to a medium comprising host cells, can provide modulation of titer of produced rAAV of different serotypes, e.g., AAV2, AAV9. In some embodiments, addition of momelotinib to a medium can provide an increased titer of produced rAAV of the AAV2 serotype as compared to a reference condition, e.g., a medium without mome...

Claims

Attorney Docket No.: 2011256-2625CLAIMS1. A method for producing a plurality of recombinant adeno-associated virus (r AAV) particles comprising:(a) transfecting one or more host cells in a culture comprising a medium with one or more vectors encoding:(i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one pay load;(ii) at least one Rep polypeptide;(iii) at least one Cap polypeptide; and(iv) at least one helper polypeptide;(b) adding an additive to the medium; and(c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles.

2. The method of claim 1 , wherein the method further comprises collecting the produced plurality of rAAV particles from the culture.

3. The method of claim 1 or 2, wherein the plurality of rAAV particles are produced at an increased titer as compared to a plurality of rAAV particles produced without the additive added to the medium.

4. The method of claim 3, wherein the increased titer is measured at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after transfection.

5. The method of claim 3 or 4, wherein the increased titer comprises or is about a 1 -fold to about a 3 -fold increased titer.

6. A method for manufacturing a pharmaceutical composition comprising a plurality of recombinant adeno-associated virus (rAAV) particles comprising:(a) transfecting one or more host cells in a culture comprising a medium with one or more vectors encoding:(i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload;(ii) at least one Rep polypeptide;Page 98 of 10712963423v 1Attorney Docket No.: 2011256-2625(iii) at least one Cap polypeptide; and(iv) at least one helper polypeptide;(b) adding an additive to the medium;(c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles;(d) collecting the produced plurality of rAAV particles from the culture; and(e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles.

7. The method of claim 6, wherein the method further comprises purifying the produced plurality of rAAV particles prior to adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers.

8. The method of any one of claims 1-7, wherein the additive comprises or is a Janus kinase (JAK) inhibitor, a phosphoinositide-dependent kinase- 1 (PDKl) / protein kinase B (AKT) signaling inhibitor, a TANK-binding kinase 1 (TBK1) inhibitor, an interferon inhibitor, an IKB kinase (IKK) inhibitor, a NF-KB inhibitor, a MNK1 inhibitor, a proteasome inhibitor, a protein neddylation inhibitor, a histone deacetylase (HDAC) inhibitor, or a protein kinase R (PKR) inhibitor or a combination thereof.

9. The method of any one of claims 1-8, wherein the additive comprises or is momelotinib, ruxolitinib, BX-795, interferon-a-IFNa-R interaction inhibitor, BMS-345541, BAY 11-7082, CGP 57380, epoxomicin, IMD 0354, imidazolo-oxindole, lactacystin, MLN-4924, NF-KB activation inhibitor, parthenolide, spermine, tosylate salt, TPCA-1, or trichostatin A, or a salt or derivative thereof.

10. The method of any one of claims 1 -9, wherein the additive comprises or is momelotinib or a salt or derivative thereof.

11. The method of claim 10, wherein momelotinib or a salt or derivative thereof is added to the medium to a concentration of about 0.5 to about 20 pM.

12. The method of claim 10, wherein momelotinib or a salt or derivative thereof and is added to the medium to a concentration of about 1.5 pM.Page 99 of 10712963423v 1Attorney Docket No.: 2011256-262513. The method of any one of claims 1-9, wherein the additive comprises or is ruxolitinib or a salt or derivative thereof.

14. The method of claim 13, wherein ruxolitinib or a salt or derivative thereof is added to the medium to a concentration of about 0.5 to about 20 pM.

15. The method of any one of claims 1-9, wherein the additive comprises or is BX-795 or a salt or derivative thereof.

16. The method of claim 15, wherein BX-795 or a salt or derivative thereof is added to the medium to a concentration of about 0.1 to about 10 pM.

17. The method of any one of claims 1-9, wherein the additive comprises or is interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof.

18. The method of claim 17, wherein interferon-a-IFNa-R interaction inhibitor or a salt or derivative thereof is added to the medium to a concentration of about 0.1 to about 25 pM.

19. The method of any one of claims 1-9, wherein the additive comprises or is BMS-345541 or a salt or derivative thereof.

20. The method of claim 19, wherein BMS-345541 or a salt or derivative thereof is added to the medium to a concentration of about 0.25 to about 25 pM.

21. The method of any one of claims 1-20, wherein the additive is added to the medium prior to transfection of the host cells with one or more vectors.

22. The method of claim 21, wherein the additive is added to the medium at about 1 to about 48 hours prior to transfection.

23. The method of any one of claims 1-22, wherein the additive is added to the medium substantially simultaneously to transfection of the host cells with one or more vectors.

24. The method of any one of claims 1-22, wherein the additive is added to the medium after transfection of the host cells with one or more vectors.Page 100 of 10712963423v 1Attorney Docket No.: 2011256-262525. The method of claim 24, wherein the additive is added to the medium at about 1 to about 60 minutes after transfection.

26. The method of any one of claims 1-25, wherein the method further comprises adding one or more additional additives.

27. The method of claim 23, wherein the one or more additional additives comprises or is a microtubule destabilizer, optionally wherein the microtubule destabilizer comprises or is a G2 / M inhibitor.

28. The method of claim 27, wherein the microtubule destabilizer comprises or is colcemid, colchicine, vinblastine, vincristine, or a salt or derivative thereof or a combination thereof.

29. The method of any one of claims 26-28, wherein the one or more additional additives ar e added to the medium prior to transfection of the host cells with one or more vectors.

30. The method of any one of claims 26-28, wherein the one or more additional additives are added to the medium substantially simultaneously to transfection of the host cells with one or more vectors.

31. The method of any one of claims 26-28, wherein the one or more additional additives ar e added to the medium after transfection of the host cells with one or more vectors.

32. The method of any one of claims 26-31 , wherein the one or more additional additives are added to the medium prior to addition of the additive.

33. The method of any one of claims 26-31, wherein the one or more additional additives ar e added to the medium substantially simultaneously to addition of the additive.

34. The method of any one of claims 26-31 , wherein the one or more additional additives are added to the medium after addition of the additive.

35. The method of any one of claims 1-34, wherein the host cells comprise or arc adherent cells.Page 101 of 10712963423v 1Attorney Docket No.: 2011256-262536. The method of any one of claims 1-34, wherein the host cells comprise or are suspension cells.

37. The method of any one of claims 1-36, wherein the host cells comprise or are mammalian cells.

38. The method of any one of claims 1-37, wherein the host cells comprise or are HEK293 cells,CHO cells, HeLa cells, or a variant thereof.

39. The method of any one of claims 1-38, wherein the one or more vectors comprises:(i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload;(ii) a second vector encoding at least one Rep polypeptide and at least one Cap polypeptide; and(iii) a third vector encoding at least one helper polypeptide.

40. The method of any one of claims 1-38, wherein the one or more vectors comprises:(i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload; and(ii) a second vector encoding at least one Rep polypeptide, at least one Cap polypeptide, and at least one helper polypeptide.

41. The method of any one of claims 1-38, wherein the one or more vectors comprises:(i) a first vector encoding at least one Cap polypeptide and at least one payload flanked by an ITR on either side of the at least one payload; and(ii) a second vector encoding at least one Rep polypeptide and at least one helper polypeptide.

42. The method of any one of claims 1 -41 , wherein the at least one helper polypeptide comprises one, two, three, or four of El , E2A, E4orf6, or VA RNA polypeptides.

43. The method of any one of claims 1-42, wherein the one or more vectors are transfected into the host cells in the presence of a transfection reagent.

44. The method of claim 43, wherein the transfection reagent comprises or is a cationic polymer, optionally wherein the cationic polymer comprises or is polyethylenimine.

45. A reaction mixture comprising:Page 102 of 10712963423v 1Attorney Docket No.: 2011256-2625(a) one or more vectors encoding:(i) at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one pay load;(ii) at least one Rep polypeptide;(iii) at least one Cap polypeptide; and(iv) at least one helper polypeptide; and(b) an additive.

46. The reaction mixture of claim 45, wherein the additive comprises or is a Janus kinase (JAK) inhibitor, a phosphoinositide-dependent kinase- 1 (PDKl)Zprotein kinase B (AKT) signaling inhibitor, a TANK-binding kinase 1 (TBK1) inhibitor, an interferon inhibitor, an IKB kinase (IKK) inhibitor, a NF-KB inhibitor, a MNK1 inhibitor, a proteasome inhibitor, a protein neddylation inhibitor, a histone deacetylase (HD AC) inhibitor, or a protein kinase R (PKR) inhibitor or a combination thereof.

47. The reaction mixture of claim 45 or 46, wherein the additive comprises or is momelotinib, ruxolitinib, BX-795, interferon-a-IFNa-R interaction inhibitor, BMS-345541, BAY 11-7082, CGP 57380, epoxomicin, IMD 0354, imidazolo-oxindole, lactacystin, MLN-4924, NF-KB activation inhibitor, parthenolide, spermine, tosylate salt, TPCA-1, or trichostatin A, or a salt or derivative thereof.

48. The reaction mixture of any one of claims 45-47, wherein the additive comprises or is momelotinib or a salt or derivative thereof.

49. The reaction mixture of any one of claims 45-47, wherein the additive comprises or is ruxolitinib or a salt or derivative thereof.

50. The reaction mixture of any one of claims 45-47, wherein the additive comprises or is BX-795 or a salt or derivative thereof.

51. The reaction mixture of any one of claims 45-47, wherein the additive comprises or is interferon- a-IFNa-R interaction inhibitor or a salt or derivative thereof.

52. The reaction mixture of any one of claims 45-47, wherein the additive comprises or is BMS- 345541 or a salt or derivative thereof.Page 103 of 10712963423v 1Attorney Docket No.: 2011256-262553. The reaction mixture of claim 45, wherein the one or more vectors comprises:(i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload;(ii) a second vector encoding at least one Rep polypeptide and at least one Cap polypeptide; and(iii) a third vector encoding at least one helper polypeptide.

54. The reaction mixture of claim 45, wherein the one or more vectors comprises:(i) a first vector encoding at least one payload flanked by an ITR on either side of the at least one payload; and(ii) a second vector encoding at least one Rep polypeptide, at least one Cap polypeptide, and at least one helper polypeptide.

55. The reaction mixture of claim 45, wherein the one or more vectors comprises:(i) a first vector encoding at least one Cap polypeptide and at least one payload flanked by an ITR on either side of the at least one payload; and(ii) a second vector encoding at least one Rep polypeptide and at least one helper polypeptide.

56. A culture comprising a plurality of host cells and a reaction mixture of any one of claims 45-55.

57. The culture of claim 56, wherein the host cells comprise or are adherent cells.

58. The culture of claim 56, wherein the host cells comprise or are suspension cells.

59. The culture of any one of claims 56-58, wherein the host cells comprise or are mammalian cells.

60. The culture of any one of claims 56-59, wherein the host cells comprise or are HEK293 cells,CHO cells, HeLa cells, or a variant thereof.

61. A bioreactor comprising a culture of any one of claims 56-60.

62. The bioreactor of claim 61, wherein the bioreactor comprises or is a batch culture bioreactor, continuous culture bioreactor, a fed batch culture bioreactor, or a perfusion culture bioreactor.Page 104 of 10712963423v 1Attorney Docket No.: 2011256-262563. A method for producing a plurality of recombinant adeno-associated virus (rAAV) particles comprising:(a) inducing expression of at least one Rep polypeptide and at least one Cap polypeptide in one or more host cells in a culture comprising a medium;(b) adding an additive to the medium; and(c) culturing the host cells under conditions suitable for production of the plurality of rAAV particles; wherein the one or more host cells are each independently a producer cell that comprises:(i) a nucleic acid sequence encoding at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload;(ii) a nucleic acid sequence encoding at least one Rep polypeptide; and(iii) a nucleic acid sequence encoding at least one Cap polypeptide.

64. The method of claim 63, wherein the method further comprises collecting the produced plurality of rAAV particles from the culture.

65. A method for manufacturing a pharmaceutical composition comprising a plurality of recombinant adeno-associated virus (rAAV) particles comprising:(a) inducing expression of at least one Rep polypeptide and at least one Cap polypeptide in one or more host cells in a culture comprising a medium;(b) adding an additive to the medium;(c) culturing the host cells under conditions suitable for production of a plurality of rAAV particles;(d) collecting the produced plurality of rAAV particles from the culture; and(e) adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers to the produced plurality of rAAV particles; wherein the one or more host cells are each independently a producer cell that comprises:(i) a nucleic acid sequence encoding at least one payload flanked by an inverted terminal repeat (ITR) on either side of the at least one payload;(ii) a nucleic acid sequence encoding at least one Rep polypeptide; and(iii) a nucleic acid sequence encoding at least one Cap polypeptide.Page 105 of 10712963423v 1Attorney Docket No.: 2011256-262566. The method of claim 65, wherein the method further comprises purifying the produced plurality of rAAV particles prior to adding one or more pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and / or pharmaceutically acceptable carriers.

67. The method of any one of claims 63-66, wherein inducing expression of at least one Rep polypeptide and at least one Cap polypeptide comprises expressing at least one helper polypeptide in the one or more host cells.

68. The method of any one of claims 63-67, wherein inducing expression of at least one Rep polypeptide and at least one Cap polypeptide comprises contacting the one or more host cells with at least one helper virus.

69. The method of any one of claims 63-68, wherein the additive comprises or is a Janus kinase (JAK) inhibitor, a phosphoinositide -dependent kinase-1 (PDKl) / protein kinase B (AKT) signaling inhibitor, a TANK-binding kinase 1 (TBK1) inhibitor, an interferon inhibitor, an IKB kinase (IKK) inhibitor, a NF-KB inhibitor, a MNK1 inhibitor, a proteasome inhibitor, a protein neddylation inhibitor, a histone deacetylase (HD AC) inhibitor, or a protein kinase R (PKR) inhibitor or a combination thereof.

70. The method of any one of claims 63-69, wherein the additive comprises or is momelotinib, ruxolitinib, BX-795, interferon-a-IFNa-R interaction inhibitor, BMS-345541, BAY 11-7082, CGP 57380, epoxomicin, IMD 0354, imidazolo-oxindole, lactacystin, MLN-4924, NF-KB activation inhibitor, parthenolide, spermine, tosylate salt, TPCA-1, or trichostatin A, or a salt or derivative thereof.

71. The method of any one of claims 63-70, wherein the additive comprises or is momelotinib or a salt or derivative thereof.Page 106 of 10712963423v 1

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