Compositions and methods for treating HER2-positive metastatic breast cancer and other cancers

Abstract

Provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising an AAV 5' inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding an anti-Her2 antibody (i.e., trastuzumab) comprising heavy and light chains operably linked to regulatory control sequences that direct expression of trastuzumab in a target cell, and an AAV 3' ITR. Also provided are pharmaceutical compositions comprising the rAAV described herein in a formulation buffer, nucleic acid molecules, packaging host cells, rAAV production systems, and methods of treating HER2-positive metastatic cancer in the brain, and optionally metastatic breast cancer in the brain.

Description

[Technical field]

[0001] Electronic Sequence Listing Reference An electronic sequence listing entitled "UPN-22-9834PCT_Seq_List.xml", created on March 30, 2023, having a size of 186,012 bytes, is filed herewith, and the contents of the electronic sequence listing (e.g., sequences and text therein) are incorporated herein by reference in their entirety. [Background technology]

[0002] Brain metastases are a common and devastating sequela of breast cancer for which treatment options are few and inadequate. 6-16% of breast cancer patients develop central nervous system (CNS) metastases. These patients have a median survival of 20% at 1 year and 1.3% at 5 years from the time of diagnosis. DiStefano A,et al.,Cancer.1979;44:1913-1918, Takakura K,et al.,Metastatic tumors of the central nervous system. Tokyo:Igaku-Shoin,1982, Hall WA,et al.,Long-term survival with metastatic cancer to the brain. Med Oncol. 2000 Nov;17(4):279-86, Pienkowski T,Zielinski CC.Trastuzumab treatment in patients with breast cancer and metastatic CNS disease.Ann Oncol.2010 May;21(5):917-24. Surgical resection of brain metastases is often infeasible, and chemotherapeutic agents are largely excluded from the CNS by the blood-brain barrier (BBB) ​​[Nakayama A,et al.,Antitumor Activity of TAK-285,an Investigational,Non-Pgp Substrate HER2 / EGFR Kinase Inhibitor,in Cultured Tumor Cells,Mouse and Rat Xenograft Tumors,and in an HER2-Positive Brain Metastasis Model,J Cancer.2013 Aug 16;4(7)]. Alternative therapies to treat breast cancer brain metastases are needed.

[0003] Breast cancers that overexpress the HER2 receptor tyrosine kinase have a high propensity to metastasize to the CNS and account for 25-30% of all breast cancer cases [Bendell JC,et al.,Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma.Cancer.2003 Jun 15;97(12):2972-7]. Trastuzumab (Herceptin®) is a first-line immunoglobulin G (IgG) monoclonal antibody (mAb) against HER2 that has been reported to significantly improve survival in patients with HER2-positive disease [Lin NU, et al., Brain metastases: the HER2 paradigm. Clin Cancer Res. 2007 Mar 15; 13(6): 1648-55; Palmieri D, et al, Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain. Cancer Res. 2007 May 1; 67(9): 4190-8]. However, patients who benefit from trastuzumab often experience concurrent CNS disease because the mAb does not cross the BBB [Nakayama, cited above]. Direct injection of trastuzumab into the CNS has been proven safe, and intrathecal administration of trastuzumab in patients with leptomeningeal carcinomatosis has been reported to increase overall survival from 2 to 13.5 months [Zagouri F,et al.,Intrathecal administration of trastuzumab for the treatment of meningeal carcinomatosis in HER2-positive metastatic breast cancer:a systematic review and pooled analysis. Breast Cancer Res Treat. 2013 May;139(1):13-22].Leptomeningeal carcinomatosis is associated with a compromised, rather than intact, blood-brain barrier. Park, EJ, et al., J Controlled Release, 163 (2012), 277-284, report that focused ultrasound bursts combined with circulating microbubbles can transiently permeabilize both the blood-brain barrier and the blood-tumor barrier to trastuzumab.

[0004] Although current therapies have led to improved control of systemic disease, treatment of metastatic dissemination of human breast cancer to the CNS remains a major therapeutic challenge. Summary of the Invention

[0005] Provided herein is a therapeutic recombinant replication-deficient adeno-associated virus (rAAV), comprising an adeno-associated virus (AAV) capsid containing a vector genome, the vector genome comprising (a) an AAV-5' inverted terminal repeat (ITR); and (b) an expression cassette comprising a coding sequence for an anti-Her2 antibody having a heavy chain and a light chain, the expression cassette comprising (i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody heavy chain, (ii) a nucleic acid sequence comprising SEQ ID NO:3, or a sequence at least 95% identical to SEQ ID NO:3, encoding the anti-Her2 antibody heavy chain, (iii) a nucleic acid sequence encoding an anti-Her2 antibody heavy chain comprising SEQ ID NO:3, or a sequence at least 95% identical to SEQ ID NO:3, (v) a furin cleavage site, (iv) a T2A element linker, (v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody light chain, (vi) a nucleic acid sequence encoding an anti-Her2 antibody light chain comprising SEQ ID NO:5, or a sequence at least 95% identical to SEQ ID NO:5, and a regulatory control sequence operably linked to a sequence in the expression cassette, the regulatory sequence comprising a ubiquitin C (UbC) promoter, an optional enhancer, an intron, and a polyadenylation (polyA) sequence, and (c) an AAV-3'ITR. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the anti-Her2 antibody heavy chain comprises SEQ ID NO:7, and the nucleic acid sequence encoding the leader peptide operably linked to the anti-Her2 antibody light chain comprises SEQ ID NO:9. In certain embodiments, the trastuzumab coding sequence comprises the nucleic acid sequence of SEQ ID NO:29, or a sequence at least 95% identical to SEQ ID NO:29. In certain embodiments, the expression cassette comprises a UbC promoter having the nucleic acid sequence of SEQ ID NO: 24. In certain embodiments, the expression cassette comprises an SV40 polyA having the nucleic acid sequence of SEQ ID NO: 23. In certain embodiments, the expression cassette comprises a chimeric intron having the nucleic acid sequence of SEQ ID NO: 22. In certain embodiments, the expression cassette comprises a nucleic acid sequence of SEQ ID NO: 26, or a nucleic acid sequence at least 99% identical to SEQ ID NO: 26. In certain embodiments, the vector genome comprises a nucleic acid sequence of SEQ ID NO: 25, or a nucleic acid sequence at least 99% identical to SEQ ID NO: 25.

[0006] In certain embodiments, provided herein is a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid containing a vector genome, the vector genome comprising (a) an AAV-5' inverted terminal repeat (ITR); and (b) an expression cassette comprising coding sequences for an anti-Her2 antibody having a heavy chain and a light chain, the expression cassette comprising (i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to the anti-Her2 antibody heavy chain, (ii) a nucleic acid sequence encoding the anti-Her2 antibody heavy chain represented by SEQ ID NO:3, or a sequence and an expression cassette comprising a nucleic acid sequence comprising a sequence at least 95% identical to SEQ ID NO:3, (iii) a Furin cleavage site, (iv) a T2A element linker, (v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody light chain, (vi) a nucleic acid sequence comprising SEQ ID NO:5, or a sequence at least 95% identical to SEQ ID NO:5, encoding an anti-Her2 antibody light chain, and a regulatory control sequence operably linked to the sequence in the expression cassette, the regulatory sequence comprising one or more of a promoter that is a chicken beta actin promoter, or a CB7 hybrid promoter comprising a chimeric intron comprising a cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta actin promoter, and a chicken beta actin intron, and a polyA sequence that is an SV40 polyA sequence. In a particular embodiment, the CB7 promoter has the nucleic acid sequence of SEQ ID NO:21. In certain embodiments, the expression cassette comprises a nucleic acid sequence of SEQ ID NO:28, a nucleic acid sequence at least 99% identical to SEQ ID NO:28, a nucleic acid sequence of SEQ ID NO:2, or a nucleic acid sequence at least 99% identical to SEQ ID NO:2. In certain embodiments, the vector genome comprises a nucleic acid sequence of SEQ ID NO:27, a sequence at least 99% identical to SEQ ID NO:27, SEQ ID NO:50, a sequence at least 99% identical to SEQ ID NO:50, SEQ ID NO:1, or a sequence at least 99% identical to SEQ ID NO:1. In certain embodiments, the capsid is an AAVhu68 capsid, an AAV9 capsid, an AAVhu95 capsid, an AAVhu96, or an AAVrh91 capsid.

[0007] In one aspect, provided herein are compositions and pharmaceutical compositions comprising the rAAV described herein and an aqueous suspension medium. In certain embodiments, the rAAV or composition thereof is for use in treating metastatic cancer in the brain, the metastatic cancer being from a HER2-positive primary tumor, optionally for use in treating metastatic breast cancer in the brain. In certain embodiments, the composition or pharmaceutical composition is formulated for central nervous system (CNS) delivery, optionally, the CNS delivery is intrathecal delivery, optionally via intracerebroventricular (ICV) injection, intracisternoma (ICM) injection, intraparenchymal injection, direct injection into the tumor or tumor bed, or via an Ommaya device. In certain embodiments, the rAAV or composition thereof is for use in preparing a medicament for the treatment of metastatic HER2-positive cancer in the brain, optionally, the metastatic HER2-positive cancer in the brain is metastatic breast cancer in the brain. In certain embodiments, the compositions may be delivered via systemic routes and / or directly to a Her2-positive tumor located outside the CNS (eg, in the breast or in a metastatic Her2-positive cancer).

[0008] In a further aspect, provided herein is a recombinant nucleic acid molecule comprising: (a) an AAV-5' inverted terminal repeat (ITR); (b) an expression cassette comprising at least one open reading frame (ORF) comprising an anti-Her2 antibody heavy chain and an anti-Her2 antibody light chain, and a nucleic acid sequence operably linked thereto that regulates expression of the anti-Her2 antibody heavy chain and the anti-Her2 antibody light chain; and (c) an AAV-3' ITR, wherein the expression cassette comprises: (i) (A) a ubiquitin C (UbC) promoter, or (B) a promoter that is a CB7 hybrid promoter comprising a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron comprising a chicken beta actin splicing donor comprising a chicken beta actin intron and a rabbit beta globin acceptor, and / or (ii) an intron that is a chimeric intron, and (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to an anti-Her2 heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3 encoding an anti-Her2 heavy chain; (v) a furin cleavage site, (vi) a T2A linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to an anti-Her2 light chain; (viii) a nucleic acid sequence comprising SEQ ID NO:5 encoding an anti-Her2 light chain; (ix) an SV40 polyadenylation (polyA) sequence, The expression cassette further comprises a spacer sequence. In certain embodiments, the nucleic acid molecule comprises an expression cassette comprising the nucleic acid sequence of SEQ ID NO: 26. In certain embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, the recombinant nucleic acid molecule is a plasmid.

[0009] In a still further aspect, there is provided herein a packaging host cell comprising a recombinant nucleic acid molecule as described herein, and further comprising an AAV rep coding sequence operably linked to a sequence that expresses the rep protein in the packaging host cell, an AAV capsid coding sequence operably linked to a sequence that expresses the AAV capsid protein in the packaging host cell, and the necessary helper virus functions to enable packaging of the expression cassette and the AAV ITRs into an AAV capsid.

[0010] In another aspect, provided herein is an rAAV production system useful for producing the rAAV described herein, the production system comprising a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein, (b) a vector genome, and (c) sufficient AAV rep and helper functions to enable packaging of the vector genome into an AAV capsid.

[0011] In yet another aspect, provided herein is a method for treating metastatic HER2-positive cancer in the brain. In certain embodiments, provided herein is a method for treating metastatic breast cancer in the brain. The method comprises administering to a subject in need thereof an effective amount of rAAV as described herein, or a composition, pharmaceutical composition, or suspension thereof. In certain embodiments, the suspension is formulated for intrathecal, intracisternal, or intraventricular administration. In certain embodiments, provided herein is an anti-neoplastic regimen comprising administering rAAV as described herein, a composition, or pharmaceutical composition thereof in combination with a biological drug, a small molecule, an anti-neoplastic agent, radiation, and / or a chemotherapeutic agent.

[0012] These and other aspects of the invention will become apparent from the following detailed description of the invention. [Brief description of the drawings]

[0013] [Figure 1A] FIG. 1 shows a schematic of the studies designed to investigate preclinical activity in mice. [Figure 1B] 1 shows results demonstrating preclinical activity of engineered trastuzumab (antitumor activity against breast cancer brain metastases) shown as plots of survival probability in mice following tumor implantation. [Figure 2A] ELISA results with plotted measurements of trastuzumab concentration (ng / mg protein) in perfused brain tissue samples collected following dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40 and AAVrh91.UbC.CI.IL2_V1_trastuzumab-coGW.SV40 (Promega Intron) in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. [Figure 2B]ELISA results with plotted measurements of trastuzumab concentration (μg / mL) in serum samples collected following dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40 and AAVrh91.UbC.CI.IL2_V1_trastuzumab-coGW.SV40 (Promega Intron) in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. The mean peak serum concentration in patients receiving 500 mg (highest dose) was 377 μg / mL. [Figure 3A] 1 shows the results of a biodistribution study with plotted measurements of DNA concentration (GC / μg) in collected liver and brain tissues. [Figure 3B] 1 shows the results of a biodistribution study with plotted measurements of RNA concentration (copies / 100 ng of RNA) in collected liver and brain tissues. [Figure 4A] Further data are shown measuring trastuzumab concentrations in serum samples collected 28 days after dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. [Figure 4B] Further data are shown measuring trastuzumab concentrations in perfused brain tissue samples collected 28 days after dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. [Figure 4C]Further data are shown measuring DNA distribution in brain and liver tissue samples collected 28 days after dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. [Figure 5A] Shown are expression levels of trastuzumab (μg / mL) measured in serum samples on days −1, 7, 14, and 28 after treatment with AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 compared to capsid control and PBS. [Figure 5B] Shown are expression levels of trastuzumab (μg / mL) measured in brain tissue samples at days −1, 7, 14, and 28 following treatment with AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 compared to capsid control and PBS. [Figure 6] Vector biodistribution (GC / diploid cells) samples are shown at days -1, 7, 14, and 28 after dosing with AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 compared to capsid control and PBS. [Figure 7] Quantification of tumor bioluminescence assessment in mouse xenografts (MDA-MB-453 (ER- / PR- / HER2+)) following treatment with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 compared to isotype control is shown. [Figure 8A] 1 shows that ICV injection of AAV resulted in sustained expression of trastuzumab in Rag1 KO mice as measured by ELISA on serum samples. [Figure 8B] We show that ICV injection of AAV resulted in sustained expression of trastuzumab in Rag1KO mice as measured using ELISA on brain homogenate (perfused) samples. [Figure 9] 1 shows Kaplan-Meier survival analysis of survival probability (disease remission) in tumor-bearing mice (MDA-MB-453 (ER- / PR- / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40. [Figure 10] Kaplan-Meier survival analysis of survival probability in tumor-bearing mice (BT-474 (ER+ / PR+ / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 (prophylactic treatment). [Figure 11A] Representative Western blots confirming expression of trastuzumab heavy chain in brain lysates following administration of AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 and AAVhu68.CMV.PI.trastuzumab.SV40 (columns 2921 and 2922) (e.g., Figures 2A and 2B). [Figure 11B] Representative Western blots confirming expression of trastuzumab light chain in brain lysates following administration of AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 and AAVhu68.CMV.PI.trastuzumab.SV40 (columns 2921 and 2922) (e.g., Figures 2A and 2B). [Figure 12A] FIG. 1 shows a schematic of the experimental design for preclinical activity studies in mice. [Figure 12B] Quantification of tumor bioluminescence assessment in mouse xenografts (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) following treatment with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 compared to isotype control is shown. [Figure 12C]1 shows a Kaplan-Meier survival analysis of survival probability in tumor-bearing mice (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40. [Figure 13A] FIG. 1 shows a schematic of the experimental design for preclinical activity studies in mice. [Figure 13B] 1 shows a Kaplan-Meier survival analysis of survival probability in tumor-bearing mice (MDA-MB-231HER2 / low tumor xenografts) treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40. [Figure 13C] HER2 expression levels in MDA-MB-231 cells as measured via flow cytometry after surface staining with an isotype control antibody are shown. [Figure 13D] HER2 expression levels in MDA-MB-231 cells as measured via flow cytometry after surface staining with HER2 antibody. [Figure 14A] FIG. 1 shows a schematic of the experimental design for preclinical activity studies in mice. [Figure 14B] Quantification of tumor burden assessment (bioluminescence assessment) in mouse xenografts (BT-474 (ER+ / PR+ / HER2+) xenografts) following treatment with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 compared to PBS and isotype controls. [Figure 14C] 1 shows a Kaplan-Meier survival analysis of survival probability in tumor-bearing mice (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenoexplants) treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40. [Figure 15A] FIG. 1 shows a schematic of the experimental design of a study examining long-term transgene expression following single or double (rechallenge) administration of AAV.trastuzumab in the MDA-MB-453 (ER- / PR- / HER2+) xenograft model. [Figure 15B]FIG. 1 shows trastuzumab expression levels measured by ELISA using serum samples collected on day 98, plotted as μg trastuzumab / mL in the MDA-MB-453 (ER- / PR- / HER2+) xenograft model. [Figure 15C] FIG. 1 shows trastuzumab expression levels measured by ELISA using brain samples (perfused brain homogenate) taken on day 98 plotted as μg trastuzumab / mL in the MDA-MB-453 (ER- / PR- / HER2+) xenograft model. [Figure 16] 1 shows trastuzumab expression levels in serum after administration of AAVhu95M199.CB7.CI.IL2_trastuzumab-coGW.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1_trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_trastuzumab-coGW.SV40 in mice. [Figure 17] FIG. 1 shows trastuzumab expression levels in the brain (perfused) following administration of AAVhu95M199.CB7.CI.IL2_trastuzumab-coGW.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1_trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_trastuzumab-coGW.SV40 in mice. [Figure 18A] A schematic diagram of the experimental design of the study to evaluate tumor burden is shown. Briefly, guide screws were implanted 7 days before cell transplantation (D-7), MDA-MB-453 cells were implanted on day 0 (2.5×105 cells / mouse), and on day 3 after cell transplantation, mice were intracranially (ICV) administered rAAV.trastuzumab at a dose of 1×1011GC / mouse, and images were taken 2, 4, and 6 weeks after cell transplantation. [Figure 18B]1 shows the results of tumor burden assessment plotted as total flux (p / s) at weeks 2, 4, and 6 after tumor cell implantation in mice treated with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. These results confirm the therapeutic efficacy of the rAAV.Trastuzumab vector. [Figure 18C] 1 shows Kaplan-Meier survival curves in mice following treatment with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. [Figure 19A] A schematic diagram is shown for the experimental design of the study to evaluate antitumor activity. Briefly, guide screws were implanted 7 days (D-7) before cell implantation, E2 pellets with BT474 clone 5 cells were implanted on day 0 (1.5 × 105 cells / mouse), and on day 3 after cell implantation, mice were administered rAAV.trastuzumab intracranially (ICV) at a dose of 1 × 1011 GC / mouse, and imaging was performed 2 weeks after cell implantation. [Figure 19B] FIG. 1 shows the results of tumor burden assessment plotted as total flux (p / s) at 2, 4, and 6 weeks post tumor cell implantation in mice treated with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. [Figure 19C]1 shows Kaplan-Meier survival curves in mice (bearing BT474 xenografts) following treatment with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. [Figure 20] 1 shows the results of a vector titration study plotted as trastuzumab expression levels measured by ELISA and mass spectrometry in mice administered rAAV.Trastuzumab at doses of 1×10 11 GC / mouse and 1×10 10 GC / mouse. [Figure 21A] FIG. 1 shows a schematic of the experimental design of the study to evaluate tumor challenge. [Figure 21B] FIG. 1 shows tumor growth plotted as total flux (p / s) measured at 2, 4, and 5 weeks after tumor implantation in mice treated with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at a dose of 1×10 GC / mouse. [Figure 22A] FIG. 1 shows a schematic of the experimental design of the study to evaluate tumor challenge. [Figure 22B] FIG. 1 shows tumor growth plotted as total flux (p / s) measured at 2, 4, and 5 weeks after tumor implantation in mice treated with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at a dose of 1×10 10 GC / mouse. [Figure 23A]1 shows the results of tumor burden assessment examined by imaging plotted as total flux (p / s) in mice administered AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at 1×10 10 GC / mouse and 1×10 11 GC / mouse. [Figure 23B] 1 shows the results of a Kaplan-Meier survival analysis plotted as the probability of survival in mice administered AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 at 1×10 10 GC / mouse and 1×10 11 GC / mouse. [Figure 24A] 1 shows trastuzumab expression levels in serum following administration of rAAV.trastuzumab with or without IVIG pretreatment. [Figure 24B] 4 shows trastuzumab expression levels in the brain (perfused) at day 30 following administration of rAAV.trastuzumab with or without IVIG pretreatment. [Figure 25A] Trastuzumab expression levels in CSF measured by ELISA at days 0.3, 7, 14, 21, and 36-37 after rAAV administration in NHPs from cohort 1b ((AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40)), cohort 2 (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40), and at day 14 in the negative control. [Figure 25B] Shown are levels of anti-drug antibodies measured in CSF samples collected in NHPs on days 0, 3, 7, 14, 21, and 36-37 after rAAV administration (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). [Figure 26A]Trastuzumab expression levels in serum measured by ELISA at days 0.3, 7, 14, 21, and 36-37 after rAAV administration in NHPs from cohort 1b ((AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40)) and cohort 2 (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). [Figure 26B] Shown are levels of anti-drug antibodies measured in serum samples collected in NHPs on days 0, 3, 7, 14, 21, and 36-37 after rAAV administration (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). [Figure 27] Quantification of trastuzumab protein in NHP brain tissue following ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). [Figure 28A] Quantification of trastuzumab protein in NHP spinal cord following ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). [Figure 28B] Quantification of trastuzumab protein in NHP dorsal root ganglia (DRG) following ICM administration of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). [Figure 29A]1 shows trastuzumab mRNA detection in NHP brain tissue (cerebellum and occipital cortex) by 10XGENOMICS (mean number of reads at final sequencing depth - 883 x 106 reads / sample). No background detection of trastuzumab was observed in untreated monkeys. 1 shows trastuzumab mRNA detection in cerebellum in NHP18-032 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. [Figure 29B] 1 shows trastuzumab mRNA detection in NHP brain tissue (cerebellum and occipital cortex) by 10XGENOMICS (mean number of reads at final sequencing depth - 883 x 106 reads / sample). No background detection of trastuzumab was observed in untreated monkeys. 1 shows trastuzumab mRNA detection in cerebellum in NHP20-198 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. [Figure 29C] 1 shows trastuzumab mRNA detection in NHP brain tissue (cerebellum and occipital cortex) by 10XGENOMICS (mean number of reads at final sequencing depth - 883 x 106 reads / sample). No background detection of trastuzumab was observed in untreated monkeys. 1 shows trastuzumab mRNA detection in the occipital cortex in NHP18-032 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. [Figure 29D] 1 shows trastuzumab mRNA detection in NHP brain tissue (cerebellum and occipital cortex) by 10XGENOMICS (mean number of reads at final sequencing depth - 883 x 106 reads / sample). No background detection of trastuzumab was observed in untreated monkeys. 1 shows trastuzumab mRNA detection in the occipital cortex in NHP20-198 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. [Figure 30A]1 shows the biodistribution of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40) in NHPs in various tissues after ICM administration. FIG shows the DNA biodistribution of AAV.Trastuzumab in NHPs in various tissues after ICM administration. [Figure 30B] 1 shows the biodistribution of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40) in NHPs in various tissues after ICM administration. 2 shows the RNA biodistribution of AAV.Trastuzumab in NHPs in various tissues after ICM administration. [Figure 31A] Figure 1 shows that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. A schematic of the AAV vector genome is shown. Data shown as individual data points and mean ± SEM. [Figure 31B] Figure 1 shows that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Schematic diagram of in vivo transgene expression assessment in healthy adult Rag1 KO female mice following ICV delivery of an AAV9 vector encoding trastuzumab (1x1011 GC / mouse). Data shown as individual data points and mean ± SEM. [Figure 31C]Figure 1 shows that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Quantification of trastuzumab by ELISA over time in serum from Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab vector (1x1011GC / mouse, n=10) or PBS (n=10). Data shown as individual data points and mean±SEM. [Figure 31D] Figure 1 shows that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Quantification of trastuzumab by ELISA over time in perfused brain tissue from Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab vector (1x1011GC / mouse, n=10) or PBS (n=10). Data shown as individual data points and mean±SEM. [Figure 31E] Figure 31C shows that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Figure 31D shows DNA biodistribution by qPCR in brain and liver tissue from Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab or PBS. Data shown as individual data points and mean ± SEM. [Fig. 31F] Figure 31C shows that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Figure 31F shows RNA biodistribution analysis by qPCR of brain and liver tissue from Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab or PBS. Data shown as individual data points and mean ± SEM. [Figure 31G]Figure 1 shows that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Western blot analysis of trastuzumab heavy and light chains in brain lysates from Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab (n=4) or PBS (n=2). Purified trastuzumab (10 ng / lane) and β-actin were used as positive and loading controls, respectively. Data shown as individual data points and mean ± SEM. [Fig. 31H] Figure 1 shows that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Figure 2 shows that trastuzumab identity was confirmed by LC-MS using brain homogenates from Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab (n=4) or PBS (n=2). Data shown as individual data points and mean ± SEM. [Figure 32A] Figure 32A shows that AAV9.UbC.Trastuzumab CNS transduction efficiency is not affected by pretreatment with intravenous immunoglobulin (IVIg) containing broadly neutralizing antibodies against AAV9. Adult female Rag1 KO mice were pretreated with IVIg or control serum from C57BL6 / J donor mice 2 hours prior to ICV administration of AAV9.UbC.Trastuzumab (1x1011GC / mouse, n=5). Mice ICV-treated with PBS (n=3) were used as controls. Figure 32A shows trastuzumab levels in serum over time from ICV-treated mice measured by ELISA. Data shown as individual data points and mean±SEM. ***p≦0.0001; ns, not significant difference. [Figure 32B]Figure 32B shows that AAV9.UbC.Trastuzumab CNS transduction efficiency is not affected by pretreatment with intravenous immunoglobulin (IVIg) containing broadly neutralizing antibodies against AAV9. Adult female Rag1 KO mice were pretreated with IVIg or control serum from C57BL6 / J donor mice 2 hours prior to ICV administration of AAV9.UbC.Trastuzumab (1x1011GC / mouse, n=5). Mice ICV-treated with PBS (n=3) were used as controls. Figure 32B shows trastuzumab levels in perfused brain tissue (day 30) from ICV-treated mice measured by ELISA. Data shown as individual data points and mean ± SEM. ***p≦0.0001; ns, not significant difference. [Figure 32C] Figure 1 shows that AAV9.UbC.Trastuzumab CNS transduction efficiency is not affected by pretreatment with intravenous immunoglobulin (IVIg) containing broadly neutralizing antibodies against AAV9. Adult female Rag1 KO mice were pretreated with IVIg or control serum from C57BL6 / J donor mice 2 hours prior to ICV administration of AAV9.UbC.Trastuzumab (1x1011GC / mouse, n=5). Mice treated ICV with PBS (n=3) were used as controls. Figure 1 shows DNA biodistribution analysis of AAV9.UbC.Trastuzumab vectors by qPCR in brain, liver, and heart 30 days after vector administration. Data shown as individual data points and mean ± SEM. ***p≦0.0001; ns, not significant difference. [Figure 33A] Figure 1 shows that single-dose ICV administration of AAV9.UbC.Trastuzumab resulted in tumor regression in a HER2+ breast brain metastasis xenograft mouse model. Three days after intracranial implantation of BT-474 (ER+ / PR+ / HER2+) or MDA-MB-453 (ER- / PR- / HER2+) human breast cancer cell lines, adult female Rag1 KO mice were injected ICV with 1x1011GC / animal of AAV9.UbC.Trastuzumab or AAV9.isotype control. Quantification of total photon flux via bioluminescence in vivo imaging system (IVIS) is shown. ***p<0.0001 [Figure 33B]Figure 1 shows that single dose ICV administration of AAV9.UbC.Trastuzumab resulted in tumor regression in a HER2+ breast brain metastasis xenograft mouse model. Three days after intracranial implantation of BT-474 (ER+ / PR+ / HER2+) or MDA-MB-453 (ER- / PR- / HER2+) human breast cancer cell lines, adult female Rag1 KO mice were injected ICV with 1x1011 GC / animal of AAV9.UbC.Trastuzumab or AAV9.Isotype control. Kaplan-Meier survival curves of mice bearing orthotopic brain tumors derived from the BT-474 cell line and treated ICV with AAV vectors (AAV9.Isotype control: n=9, or AAV9.UbC.Trastuzumab: n=12). ***p<0.0001 [Figure 33C] Figure 1 shows that single-dose ICV administration of AAV9.UbC.Trastuzumab resulted in tumor regression in a HER2+ breast brain metastasis xenograft mouse model. Three days after intracranial implantation of BT-474 (ER+ / PR+ / HER2+) or MDA-MB-453 (ER- / PR- / HER2+) human breast cancer cell lines, adult female Rag1 KO mice were injected ICV with 1x1011GC / animal of AAV9.UbC.Trastuzumab or AAV9.isotype control. Representative bioluminescence IVIS images (week 4, left panel), total photon flux signal quantification (right panel) are shown. ***p<0.0001 [Figure 33D] Figure 1 shows that single dose ICV administration of AAV9.UbC.Trastuzumab resulted in tumor regression in a HER2+ breast brain metastasis xenograft mouse model. Three days after intracranial implantation of BT-474 (ER+ / PR+ / HER2+) or MDA-MB-453 (ER- / PR- / HER2+) human breast cancer cell lines, adult female Rag1 KO mice were injected ICV with 1x1011GC / animal of AAV9.UbC.Trastuzumab or AAV9.Isotype control. Kaplan-Meier survival curves of mice bearing orthotopic brain tumors derived from MDA-MB-453 cell line after ICV treatment (AAV9.Isotype control: n=8, or AAV9.UbC.Trastuzumab: n=7) are shown. ***p<0.0001 [Figure 33E]Figure 33C shows that single-dose ICV administration of AAV9.UbC.Trastuzumab resulted in tumor regression in a HER2+ breast brain metastasis xenograft mouse model. Three days after intracranial implantation of BT-474 (ER+ / PR+ / HER2+) or MDA-MB-453 (ER- / PR- / HER2+) human breast cancer cell lines, adult female Rag1 KO mice were injected ICV with 1x1011 GC / animal of AAV9.UbC.Trastuzumab or AAV9.isotype control. Figure 33C shows long-term trastuzumab expression quantified in perfused brain tissue by ELISA in a subset of mice shown in Figure 33D (n=5) 104 days after ICV administration of AAV9.UbC.Trastuzumab. ***p<0.0001 [Figure 34A] Figure 34 shows detection of trastuzumab in the CSF of female NHPs after intracisternal (ICM) administration of an AAV9 vector encoding trastuzumab. Schematic design of experimental ICM delivery of AAV9.UbC.Trastuzumab (n=2) vectors at 3x1013GC / animal in NHPs is shown, informing the in-life blood and CSF collection schedules and planned study end-points. NHP No. 3 was ICM injected with 3x1013GC AAV9 vector expressing GFP and was used as a negative control. Detection of trastuzumab, a humanized IgG1 antibody, in the CSF (Figure 34B) and serum (Figure 34C) of treated animals was determined by ELISA after quantification of human IgG and anti-drug antibodies against trastuzumab in the CSF (Figure 34D) and serum (Figure 34E). [Figure 34B] Figure 34 shows detection of trastuzumab in the CSF of female NHPs after intracisternal (ICM) administration of an AAV9 vector encoding trastuzumab. NHP No. 3 was ICM injected with 3x1013GC AAV9 vector expressing GFP and used as a negative control. Detection of trastuzumab, a humanized IgG1 antibody, in the CSF (Figure 34B) and serum (Figure 34C) of treated animals was determined by ELISA after quantification of human IgG and anti-drug antibodies against trastuzumab in the CSF (Figure 34D) and serum (Figure 34E). [Figure 34C]Figure 34 shows detection of trastuzumab in the CSF of female NHPs after intracisternal (ICM) administration of an AAV9 vector encoding trastuzumab. NHP No. 3 was ICM injected with 3x1013GC AAV9 vector expressing GFP and used as a negative control. Detection of trastuzumab, a humanized IgG1 antibody, in the CSF (Figure 34B) and serum (Figure 34C) of treated animals was determined by ELISA after quantification of human IgG and anti-drug antibodies against trastuzumab in the CSF (Figure 34D) and serum (Figure 34E). [Fig. 34D] Figure 34 shows detection of trastuzumab in the CSF of female NHPs after intracisternal (ICM) administration of an AAV9 vector encoding trastuzumab. NHP No. 3 was ICM injected with 3x1013GC AAV9 vector expressing GFP and used as a negative control. Detection of trastuzumab, a humanized IgG1 antibody, in the CSF (Figure 34B) and serum (Figure 34C) of treated animals was determined by ELISA after quantification of human IgG and anti-drug antibodies against trastuzumab in the CSF (Figure 34D) and serum (Figure 34E). [Figure 34E] Figure 34 shows detection of trastuzumab in the CSF of female NHPs after intracisternal (ICM) administration of an AAV9 vector encoding trastuzumab. NHP No. 3 was ICM injected with 3x1013GC AAV9 vector expressing GFP and used as a negative control. Detection of trastuzumab, a humanized IgG1 antibody, in the CSF (Figure 34B) and serum (Figure 34C) of treated animals was determined by ELISA after quantification of human IgG and anti-drug antibodies against trastuzumab in the CSF (Figure 34D) and serum (Figure 34E). [Figure 35A] Figure 1 shows gene delivery and transgene transcription throughout the CNS following ICM treatment with AAV9.UbC.Trastuzumab at 3x1013 GC / animal. Absolute quantification of AAV vector GC in NHPs 36-37 days following ICM administration of AAV9.UbC.Trastuzumab (n=2) vector. [Figure 35B]Gene delivery and transgene transcription throughout the CNS following ICM treatment with AAV9.UbC.Trastuzumab at 3x1013 GC / animal. Transcripts by qPCR in selected tissues in NHPs 36-37 days after ICM administration of AAV9.UbC.Trastuzumab (n=2) vectors. [Figure 36A] 1 shows gene delivery and transgene transcription throughout the CNS following ICM treatment with AAV9.UbC.Trastuzumab at 3×10 13 GC / animal. [Figure 36B] 1 shows gene delivery and transgene transcription throughout the CNS following ICM treatment with AAV9.UbC.Trastuzumab at 3×10 13 GC / animal. [Figure 36C] 1 shows gene delivery and transgene transcription throughout the CNS following ICM treatment with AAV9.UbC.Trastuzumab at 3×10 13 GC / animal. [Figure 36D] 1 shows gene delivery and transgene transcription throughout the CNS following ICM treatment with AAV9.UbC.Trastuzumab at 3×10 13 GC / animal. [Figure 36A] 13 shows the results of single nucleus RNA sequencing demonstrating the presence of trastuzumab mRNA transcripts in the cerebellum and occipital cortex from animals ICM-injected with the AAV9.UbC.Trastuzumab vector. [Figure 36B] 13 shows the results of single nucleus RNA sequencing demonstrating the presence of trastuzumab mRNA transcripts in the cerebellum and occipital cortex from animals ICM-injected with the AAV9.UbC.Trastuzumab vector. [Figure 36C] 13 shows the results of single nucleus RNA sequencing demonstrating the presence of trastuzumab mRNA transcripts in the cerebellum and occipital cortex from animals ICM-injected with the AAV9.UbC.Trastuzumab vector. [Figure 36D] 13 shows the results of single nucleus RNA sequencing demonstrating the presence of trastuzumab mRNA transcripts in the cerebellum and occipital cortex from animals ICM-injected with the AAV9.UbC.Trastuzumab vector. [Figure 37A]Figure 1 shows that ICM delivery of 3x1013GC / animal with AAV9.UbC.Trastuzumab results in transgene protein expression in NHP CNS tissues at levels sufficient to induce a complete anti-tumor response in tumor-bearing mice. Experimental scheme shown. Results of LC-MS analysis detecting unique AAV-encoded trastuzumab-derived peptide (DTYIHWVR, SEQ ID NO:56) in NHP brain homogenates. Trastuzumab levels expressed as fold change relative to input reference positive control (10 ng trastuzumab). ***p<0.0001 [Figure 37B] Figure 1 shows that ICM delivery of 3x1013 GC / animal with AAV9.UbC.Trastuzumab results in transgene protein expression in NHP CNS tissue at levels sufficient to induce a complete anti-tumor response in tumor-bearing mice. Figure 2 shows expression of trastuzumab in perfused brain tissue from female Rag1 KO mice 28 days after ICV administration of 1x1010 GC / animal with AAV9.isotype control (n=8) or AAV9.UbC.Trastuzumab (n=8) as measured by ELISA. ***p<0.0001 [Figure 37C] Figure 1 shows that ICM delivery of 3x1013GC / animal with AAV9.UbC.Trastuzumab results in transgene protein expression in NHP CNS tissues at levels sufficient to induce a complete anti-tumor response in tumor-bearing mice. Figure 2 shows trastuzumab protein expression in perfused brain regions by ELISA by determining tissue levels of human IgG. ***p<0.0001 [Figure 37D] Figure 1 shows that ICM delivery of 3x1013 GC / animal with AAV9.UbC.Trastuzumab results in transgene protein expression in NHP CNS tissues at levels sufficient to induce a complete anti-tumor response in tumor-bearing mice. Quantification of total photon flux by IVIS imaging of adult female Rag1 KO mice treated ICV with 1x1010 GC / animal AAV9.isotype control (n=8) or AAV9.UbC.Trastuzumab (n=8) 3 days after intracranial tumor cell implantation of reporter MDA MB453 breast cancer cells. ***p<0.0001 [Figure 37E] Figure 1 shows that ICM delivery of 3x1013GC / animal with AAV9.UbC.Trastuzumab results in transgene protein expression in NHP CNS tissues at levels sufficient to induce a complete anti-tumor response in tumor-bearing mice. Figure 2 shows Trastuzumab protein expression in the spinal cord quantified by ELISA by determining tissue levels of human IgG. ***p<0.0001 [Figure 37F] Figure 1 shows that ICM delivery of 3x1013 GC / animal with AAV9.UbC.Trastuzumab results in transgene protein expression in NHP CNS tissues at levels sufficient to induce a complete anti-tumor response in tumor-bearing mice. Kaplan-Meier survival curves of adult female Rag1 KO mice treated ICV with AAV9.isotype control (n=8) or AAV9.UbC.Trastuzumab (n=8) at 1x1010 GC / animal 3 days after intracranial tumor cell implantation of reporter MDA MB453 breast cancer cells. ***p<0.0001 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Provided herein are recombinant, replication-deficient adeno-associated virus (rAAV) vectors having an AAV capsid and packaging therein a vector genome comprising at least one open reading frame comprising engineered nucleic acid sequences encoding the heavy and light chains of an anti-HER2 antibody (e.g., antineoplastic immunoglobulin construct, trastuzumab), as well as compositions containing the same suitable for administration for the treatment of anti-Her2 cancers, including metastatic cancers. Various methods of administration are provided, including systemic administration, direct administration (e.g., intratumoral) from a primary tumor to a secondary tumor (metastatic cancer), and administration (e.g., intrathecal) to the central nervous system, particularly for the treatment of metastatic Her2-positive cancers. Also provided are pharmaceutical compositions, formulations, and particularly liquid aqueous suspensions containing the same. Uses of these compositions are also provided. Also provided are methods of compositions useful for the treatment and / or prevention of metastatic HER2-positive (HER2-human epidermal growth factor receptor 2, also referred to as Her2-positive, HER2+, or Her2+) cancers, for example in the brain.

[0015] In certain embodiments, the compositions and regimens described herein are useful for delivery of anti-neoplastic immunoglobulin constructs to the central nervous system. The compositions described herein, including AAV-Ig, are highly suitable for central nervous system (CNS) HER2-positive cancers (tumors), especially those located in the brain. The compositions and regimens are also useful for treating primary and / or secondary Her2-positive breast cancer, primary and / or secondary Her2-positive gastric and / or primary and / or secondary Her2-positive gastric gastroesophageal junction cancer, and other HER-2-positive solid tumors and cancers.

[0016] As used herein, the term "CNS neoplasm" includes primary or metastatic cancers that may be located in the brain (intracranial), meninges (connective tissue layers covering the brain and spinal cord), or spinal cord. Examples of primary CNS cancers may be, among others, gliomas (which may include glioblastomas (also known as glioblastoma multiforme), astrocytomas, oligodendrogliomas, and ependymomas, as well as mixed gliomas), meningiomas, medulloblastomas, neuromas, and primary CNS lymphomas (in the brain, spinal cord, or meninges). Examples of metastatic cancers include those originating from another tissue or organ, such as breast, lung, lymphoma, leukemia, melanoma (skin cancer), colon, kidney, prostate, or other types that metastasize to the brain.

[0017] As used herein, an "antineoplastic" immunoglobulin construct (including an antibody or antibody fragment as defined herein) encodes a polypeptide-based moiety that binds to a cell surface antigen or receptor located on a cancer cell or solid tumor, inhibits or prevents the growth and spread of tumor or malignant cells in non-solid tumors, and optionally reduces the size of the tumor. Anti-neoplastic immunoglobulin polypeptides can function by several mechanisms, for example, by inhibiting tumor cell growth by blocking growth factor receptors, cross-linking cell membrane antigens to deliver signals that control the cell cycle, blocking angiogenesis, blocking DNA repair after chemotherapy, or even inducing cell death. Alternatively, they can indirectly affect tumor growth by activating host immune effector functions such as antibody-dependent and complement-mediated cytotoxicity. In one embodiment, the antineoplastic effect of the compositions and regimens described herein can be measured by a reduction in tumor size and / or an increase in progression-free survival compared to subjects that are untreated or treated with other regimens.

[0018] The term "immunoglobulin" is used herein to include antibodies, functional fragments thereof, and immunoadhesins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, intrabodies ("intrabodies"), recombinant antibodies, multispecific antibodies, antibody fragments such as Fv, Fab, F(ab)2, F(ab)3, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem / bis-scFv, Fc, pFc', scFvFc (or scFv-Fc), disulfide Fv (dsfv), bispecific antibodies (bc-scFv) such as BiTE antibodies, camelid antibodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as NANOBODY®), chimeric antibodies, chimeric antibodies comprising at least one human constant region, and the like. "Antibody fragment" refers to at least a portion of the variable region of an immunoglobulin that binds to its target, eg, a tumor cell, a tumor cell receptor.

[0019] The rAAV vector encodes an anti-HER2 antibody (e.g., trastuzumab) heavy and light chains as described herein. See, e.g., drugbank.ca / drugs / DB00072. The encoded trastuzumab heavy chain amino acid sequence is: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35, drugbank.ca / drugs / DB00072).In certain embodiments, the encoded trastuzumab heavy chain amino acid sequence is: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 4, i.e., without the terminal lysine (K)).

[0020] The trastuzumab light chain binds to the extracellular domain of the human epidermal growth factor receptor protein with high affinity (K d=5 nM). The encoded amino acid sequence of the trastuzumab light chain is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 36, (drugbank_ca / drugs / DB00072), also SEQ ID NO: 6), which sequences are incorporated herein by reference. See also 212-Pb-TCMC-Trastuzumab [Areva Med, Bethesda, MD]. As described herein, a series of novel rAAV.anti-HER2-antibody (rAAV.trastuzumab or AAV.trastuzumab) constructs have been developed that have demonstrated high yield, expression levels, and / or activity. In certain embodiments, the size of the antibody (protein) chains can be confirmed by Western blot. In certain embodiments, the size of the antibody (protein) chains can be confirmed by mass spectrometry.

[0021] Provided herein are coding sequence(s), expression cassette(s), and vector genome(s) encoding the trastuzumab heavy chain (HC) amino acid sequence of SEQ ID NO: 4 (amino acids 1-449 of SEQ ID NO: 35) and the light chain (LC) amino acid sequence of SEQ ID NO: 6 (also SEQ ID NO: 36), each engineered to further have an exogenous leader sequence (also referred to as a "leader peptide" or "signal peptide" or "signal sequence") for each heavy and light chain (e.g., SEQ ID NO: 14 for the heavy chain amino acid sequence including the leader peptide, and SEQ ID NO: 16 for the light chain amino acid sequence including the leader peptide). In certain constructs provided herein, the leader sequence is derived from the human interleukin 2 (IL2 or IL-2) leader. In certain embodiments, the IL2 is the same for each heavy and light chain. In certain embodiments, the IL2 is a modified IL2 in at least one of the heavy and light chains. Furthermore, in certain constructs shown in the working examples, the heavy and light chains are separated by a T2A linker, which may result in one or more additional amino acids being added to the heavy chain [SEQ ID NO: 4 or SEQ ID NO: 14 (including leader peptide)]. Furthermore, in certain constructs shown in the working examples, the heavy and light chains are separated by a Furin / T2A linker, which may result in one or more additional amino acids being added to the heavy chain [SEQ ID NO: 4 or SEQ ID NO: 14 (including leader peptide)]. In one embodiment, a single arginine [R] is added to the heavy chain. However, in certain embodiments, a different linker may be selected and / or a different system may result in no additional amino acid or in one or more additional amino acids [e.g., R, Lys(K), RK, RKR, RKRR (SEQ ID NO: 49), among others]. In constructs encoding the anti-Her2 antibody trastuzumab product, the various designations following the term trastuzumab, e.g., trastuzumab-coGW, trastuzumab-coX, trastuzumab-coGY, trastuzumab, refer to different nucleic acid coding sequences in the open reading frames of the anti-Her2 antibody heavy and light chains.After cleavage of the leader peptide and assembly of the anti-Her2 light and heavy chains, optionally with mutated amino acids, the resulting anti-Her2 antibody product, alternatively referred to herein as "anti-Her2 antibody" or trastuzumab, will be understood to include variant or mutant amino acid sequences compared to the amino acid sequences of SEQ ID NO: 35 (drugbank.ca / drugs / DB00072, trastuzumab heavy chain) and SEQ ID NO: 36 (drugbank_ca / drugs / DB00072, trastuzumab light chain). In certain embodiments, the anti-Her2 antibody may include one or more conservative, non-conservative amino acid substitutions, as well as insertions, truncations, and / or deletions, compared to the amino acid sequences of SEQ ID NO: 35 (drugbank.ca / drugs / DB00072, trastuzumab heavy chain) and SEQ ID NO: 36 (drugbank_ca / drugs / DB00072, trastuzumab light chain). In certain embodiments, the amino acid sequence of the anti-Her2 antibody comprises a truncation of a terminal lysine (K) at the C-terminus of the amino acid sequence compared to SEQ ID NO:35.

[0022] In certain embodiments, the encoded amino acid sequence of trastuzumab has 724 amino acids and includes trastuzumab heavy and light chains separated by additional amino acids as a result of the linker. For example, each of the expression cassettes described herein encodes the same trastuzumab heavy and light chains, but in one embodiment, one amino acid may be added to the last position of the heavy chain. In yet other embodiments, there may be two, three, four, or more additional amino acids attached to the heavy chain. For example, in certain embodiments, the nucleic acid sequences encoding the heavy and light chains of trastuzumab are separated by a self-cleaving furin / T2A linker. A furin recognition site consisting of an arginine-lysine-arginine-arginine amino acid sequence may be used. Due to the mechanism of furin-mediated cleavage, vector-expressed trastuzumab may include an additional arginine (R) residue added to the last position of the heavy chain [SEQ ID NO: 4 or SEQ ID NO: 14 (including the leader peptide)]. In other embodiments, the vector-expressed trastuzumab is modified to include the dipeptide arginine-lysine at the end of the heavy chain, the tripeptide arginine-lysine-arginine at the end of the heavy chain, or the polypeptide arginine-lysine-arginine-arginine at the end of the heavy chain. In certain embodiments, the vector-expressed trastuzumab immunoglobulin is a heterogeneous mixture of two or more of these immunoglobulin products. Other furin cleavage sites (arginine-XX-arginine, or arginine-X-lysine or arginine-arginine) can be used, which can also generate C-terminal heterogeneity. In other words, the other vector-expressed trastuzumab immunoglobulin can be a heterogeneous population of immunoglobulins in which the heavy chain has 0, 1, 2, 3, or 4 amino acids at its C-terminus as a result of linker processing.

[0023] In addition, the light and heavy chains each contain a heterologous leader peptide that directs each of the immunoglobulin chains to the appropriate cellular compartment where the leader polypeptide is processed by the host cell machinery away from the mature immunoglobulin chain, e.g., the HC with leader peptide comprises the amino acid sequence of SEQ ID NO: 14 and the LC with leader peptide comprises the amino acid sequence of SEQ ID NO: 16, enabling the two chains to self-assemble into a recombinant anti-Her antibody in vivo.

[0024] In certain embodiments, in vivo self-assembly into recombinant trastuzumab antibodies can be confirmed by native gel electrophoresis (e.g., polyacrylamide gel electrophoresis (native-PAGE)) and denaturing gel electrophoresis (i.e., SDS-PAGE). In certain embodiments, trastuzumab does not contain the HC or LC leader peptide sequences (the HC amino acid sequence of SEQ ID NO:4 and the LC amino acid sequence of SEQ ID NO:6).

[0025] As used herein, the term "trastuzumab" refers to a heavy chain having an amino acid sequence of SEQ ID NO:4, or a sequence that is about 95% to about 100% identical thereto, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.9%, and values ​​therebetween (e.g., the heavy chain has -1, 0, 1, 2, 3, or 4 amino acids at its C-terminus as a result of linker processing), when determined over a contiguous amino acid sequence, and an amino acid sequence of SEQ ID NO:6, or a sequence that is about 95% to about 100% identical thereto, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.9%, and values ​​therebetween. "anti-Her2 antibody" refers to an immunoglobulin construct ("anti-Her2 antibody") that includes a light chain having a sequence that is about 99% to about 100% identical thereto, or at least 99.9% identical thereto, and values ​​therebetween, (e.g., as a result of linker processing) and provides at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or similar and / or identical, or greater than 100% of the biological activity (e.g., binding) or function of trastuzumab immunoglobulin product. In certain embodiments, when produced in vitro (e.g., in CHO cells), greater than 100%, e.g., about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, or more of the activity and / or function of trastuzumab is achieved. This biological activity or function may be determined by any suitable means, such as in vitro assays, animal models, or by monitoring patients after treatment. In certain embodiments, the AAV-expressed trastuzumab immunoglobulin heavy and light chains provide advantages over protein-based trastuzumab compositions derived from in vitro produced trastuzumab, including, for example, improved glycosylation, reduced deamidation, and / or improved stability.

[0026] In one embodiment, the engineered coding sequence for the heavy and light chains of trastuzumab is provided in SEQ ID NO: 17. In one embodiment, the engineered coding sequence for the heavy and light chains of trastuzumab is provided in SEQ ID NO: 29. In a particular embodiment, the present invention provides at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 3 encoding a trastuzumab heavy chain, or a sequence at least 95% identical to SEQ ID NO: 3, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, and a nucleic acid sequence comprising SEQ ID NO: 5 encoding a trastuzumab light chain, or a sequence at least 95% identical to SEQ ID NO: 5. In certain embodiments, the present invention provides at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 3 encoding the trastuzumab heavy chain, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, and a nucleic acid sequence comprising SEQ ID NO: 5 encoding the trastuzumab light chain. In certain embodiments, the linker between the heavy and light chains is a Thosea asigna virus (T2A) linker. In certain embodiments, the T2A linker comprises a nucleic acid sequence of SEQ ID NO: 32, or a sequence at least 95% identical to SEQ ID NO: 32. In certain embodiments, the T2A linker comprises a nucleic acid sequence of SEQ ID NO: 32 encoding an amino acid sequence of SEQ ID NO: 52, or a sequence at least 95% identical to SEQ ID NO: 32. In certain embodiments, the linker further comprises a furin cleavage at the 5' end of T2A, optionally connected via a flexible linker (e.g., a "GSG linker"). In certain embodiments, the Furin cleavage sequence comprises the nucleic acid sequence of SEQ ID NO: 31, or a sequence at least 95% identical to SEQ ID NO: 31. In certain embodiments, the Furin / T2A linker has the nucleic acid sequence of SEQ ID NO: 33, or a sequence at least 95% identical to SEQ ID NO: 33. In certain embodiments, the Furin / T2A linker comprises the nucleic acid sequence of SEQ ID NO: 33, which encodes the amino acid sequence of SEQ ID NO: 53, or a sequence at least 95% identical to SEQ ID NO: 33.In a particular embodiment, the present invention provides at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 3 encoding the trastuzumab heavy chain, a linker comprising a furin cleavage site and T2A, the furin cleavage site being at the 5' end of T2A, optionally connected via a flexible linker (e.g., a "GSG linker"), a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, and a nucleic acid sequence comprising SEQ ID NO: 5 encoding the trastuzumab light chain. In a particular embodiment, the trastuzumab heavy chain has an amino acid sequence of SEQ ID NO: 4 together with the leader sequence. In another embodiment, the trastuzumab light chain has an amino acid sequence of SEQ ID NO: 6 together with the leader sequence. For example, the leader sequence can be from about 15 to about 25 amino acids, preferably about 20 amino acids. In a particular embodiment, the processing of the trastuzumab heavy and light chains is directed by a leader peptide derived from the human IL2 protein. In one embodiment, the leader sequence is an interleukin (IL) IL-2 leader sequence, which may be wild type human IL2, MYRMQLLSCIALSLALVTNS [SEQ ID NO:8], or a mutated leader such as MYRMQLLLLIALSLALVTNS [SEQ ID NO:10] or MRMQLLLLIALSLALVTNS [SEQ ID NO:12]. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO:7, or a sequence at least about 95% identical to SEQ ID NO:7. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO:7 that encodes SEQ ID NO:8, or a sequence at least about 95% identical to SEQ ID NO:7. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO:9, or a sequence at least about 95% identical to SEQ ID NO:9. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO:9 that encodes SEQ ID NO:10, or a sequence at least about 95% identical to SEQ ID NO:9. In certain embodiments, the leader peptide comprises a nucleic acid sequence comprising SEQ ID NO:11, or a sequence at least about 95% identical to SEQ ID NO:11.In certain embodiments, the leader peptide comprises SEQ ID NO:11, which encodes SEQ ID NO:12, or a nucleic acid sequence comprising a sequence at least 95% identical to SEQ ID NO:11. In certain embodiments, the processing of the trastuzumab heavy and light chains is directed by leader peptides that are the same. In certain embodiments, the processing of the trastuzumab heavy and light chains is directed by different leader peptides. In certain embodiments, the trastuzumab heavy chain is directed by a leader peptide comprising the amino acid sequence of SEQ ID NO:8. In certain embodiments, the trastuzumab heavy chain is directed by a leader peptide comprising the amino acid sequence of SEQ ID NO:8 encoded by the nucleic acid sequence of SEQ ID NO:7. In certain embodiments, the trastuzumab light chain is directed by a leader peptide comprising the amino acid sequence of SEQ ID NO:10. In certain embodiments, the trastuzumab light chain is directed by a leader peptide comprising the amino acid sequence of SEQ ID NO:10 encoded by the nucleic acid sequence of SEQ ID NO:9. In certain embodiments, the trastuzumab light chain is directed by a leader peptide comprising the amino acid sequence of SEQ ID NO:12. In a specific embodiment, the trastuzumab light chain is directed by a leader peptide comprising the amino acid sequence of SEQ ID NO: 12, which is encoded by the nucleic acid sequence of SEQ ID NO: 11. Other leader sequences, or other leaders exogenous to the heavy and light chains, can be used.

[0027] In certain embodiments, provided herein is at least one ORF comprising: a nucleic acid sequence comprising SEQ ID NO:7 encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain; a linker comprising a furin cleavage site (SEQ ID NO:31), a "GSG" linker, and T2A (SEQ ID NO:32), wherein the furin cleavage site is at the 5' end of T2A; a nucleic acid sequence comprising SEQ ID NO:9 encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain.

[0028] In certain embodiments, provided herein is at least one ORF comprising: a nucleic acid sequence comprising SEQ ID NO:7 encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain; a linker comprising a furin cleavage site (SEQ ID NO:31), a "GSG" linker, and T2A (SEQ ID NO:32), wherein the furin cleavage site is at the 5' end of T2A; a nucleic acid sequence comprising SEQ ID NO:11 encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain.

[0029] As used herein, the term "biological sample" refers to any cell, biological fluid or tissue. Samples suitable for use in the present invention may include, but are not limited to, whole blood, white blood cells, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid, and skin cells. Such samples may be further diluted with saline, buffer, or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means.

[0030] As used herein, "patient" or "subject" refers to male or female humans, dogs, and animal models used for clinical research. In one embodiment, the subject of these methods and compositions is a human diagnosed with metastatic HER2 positive (HER2+) cancer in the brain, optionally, the metastatic HER2+ cancer in the brain is breast cancer, and optionally, the metastatic HER2+ cancer in the brain is gastrointestinal cancer. In certain embodiments, the human subject of these methods and compositions is a prenatal, neonatal, infant, toddler, preschooler, elementary school student, teen, young adult, or adult.

[0031] With respect to these descriptions of the invention, each of the compositions described herein is contemplated, in separate embodiments, to be useful in the methods of the invention. In addition, each of the compositions described herein that are useful in the methods is also contemplated, in separate embodiments, to be an embodiment of the invention itself.

[0032] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published documents which provide such persons with general guidance to many of the terms used in this application.

[0033] Open Reading Frames and Nucleic Acid Molecules In one aspect, an engineered nucleic acid sequence is provided that encodes an anti-Her2 antibody (i.e., a trastuzumab immunoglobulin) (also referred to as trastuzumab-coGW). The trastuzumab immunoglobulin comprises a heavy chain and a light chain. In one embodiment, the engineered nucleic acid sequence is useful for improving production, transcription, expression, or safety in a subject. In another embodiment, the engineered sequence is useful for increasing the efficacy of the resulting therapeutic composition or treatment. In a further embodiment, the engineered sequence is useful for increasing the efficacy of the expressed trastuzumab immunoglobulin, but may allow for lowering the dose of the therapeutic reagent that delivers the immunoglobulin to increase safety. In certain embodiments, provided herein is a recombinant nucleic acid molecule that comprises a nucleic acid sequence that encodes a trastuzumab immunoglobulin. In certain embodiments, the recombinant nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 17, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and values ​​therebetween that are identical to SEQ ID NO: 17. In certain embodiments, the recombinant nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 17. In certain embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 29, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and any value therebetween that is identical to SEQ ID NO: 29. In certain embodiments, the recombinant nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 29.

[0034] In one embodiment, the engineered coding sequence for the heavy and light chains of trastuzumab is provided in SEQ ID NO: 17. In certain embodiments, the engineered coding sequence for the heavy and light chains of trastuzumab comprises a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and any values ​​therebetween identical to SEQ ID NO: 17. In one embodiment, the engineered coding sequence for the heavy and light chains of trastuzumab is provided in SEQ ID NO: 29. In certain embodiments, the engineered coding sequence for the heavy and light chains of trastuzumab comprises a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and any values ​​therebetween identical to SEQ ID NO: 29. In certain embodiments, the present invention provides at least one open reading frame (ORF) comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, a nucleic acid sequence encoding the trastuzumab heavy chain comprising SEQ ID NO: 3, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, and a nucleic acid sequence encoding the trastuzumab light chain comprising SEQ ID NO: 5. In certain embodiments, the nucleic acid sequence encoding the trastuzumab heavy chain comprises SEQ ID NO: 3, or a sequence identical to SEQ ID NO: 3, including at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and values ​​therebetween. In certain embodiments, the nucleic acid sequence encoding the trastuzumab light chain comprises SEQ ID NO: 3, or a sequence identical to SEQ ID NO: 5, including at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and values ​​therebetween.In certain embodiments, the present invention provides at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 3 encoding a trastuzumab heavy chain comprising the amino acid sequence of SEQ ID NO: 4, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, and a nucleic acid sequence comprising SEQ ID NO: 5 encoding a trastuzumab light chain comprising the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the present invention provides at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, SEQ ID NO: 3 encoding a trastuzumab heavy chain comprising the amino acid sequence of SEQ ID NO: 4, or a sequence at least 95% identical to SEQ ID NO: 3, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, and a nucleic acid sequence comprising SEQ ID NO: 5 encoding a trastuzumab light chain comprising the amino acid sequence of SEQ ID NO: 6, or a sequence at least 95% identical to SEQ ID NO: 5.

[0035] In certain embodiments, the linker between the heavy and light chains is a Tosea asignavirus (T2A) linker. In certain embodiments, the T2A linker has a nucleic acid sequence of SEQ ID NO:32, or a sequence at least 95% identical to SEQ ID NO:32. In certain embodiments, the linker further comprises a furin cleavage at the 5' end of T2A, optionally connected via a flexible linker (e.g., a "GSG linker"). In certain embodiments, the furin cleavage sequence comprises a nucleic acid sequence of SEQ ID NO:31, or a sequence at least 95% identical to SEQ ID NO:31. In certain embodiments, the furin / T2A linker has a nucleic acid sequence of SEQ ID NO:33, or a sequence at least 95% identical to SEQ ID NO:33.

[0036] In certain embodiments, the leader peptide is an interleukin (IL) IL-2 leader peptide. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the trastuzumab heavy chain comprises the nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the trastuzumab heavy chain comprises the nucleic acid sequence of SEQ ID NO: 7, which encodes the amino acid sequence of SEQ ID NO: 8, which is an IL2 leader peptide. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the trastuzumab heavy chain comprises the nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the trastuzumab heavy chain comprises the nucleic acid sequence of SEQ ID NO: 9, which encodes the amino acid sequence of SEQ ID NO: 10, which is a modified IL2 leader peptide. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the trastuzumab heavy chain comprises the nucleic acid sequence of SEQ ID NO: 11. In certain embodiments, the nucleic acid sequence encoding the leader peptide operably linked to the trastuzumab heavy chain comprises the nucleic acid sequence of SEQ ID NO: 11, which encodes the amino acid sequence of SEQ ID NO: 12, which is a modified IL2 leader peptide. In certain embodiments, the nucleic acid sequence encoding a leader peptide operably linked to the trastuzumab light chain comprises the nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the nucleic acid sequence encoding a leader peptide operably linked to the trastuzumab light chain comprises the nucleic acid sequence of SEQ ID NO: 7, which encodes the amino acid sequence of SEQ ID NO: 8, which is an IL2 leader peptide. In certain embodiments, the nucleic acid sequence encoding a leader peptide operably linked to the trastuzumab light chain comprises the nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the nucleic acid sequence encoding a leader peptide operably linked to the trastuzumab light chain comprises the nucleic acid sequence of SEQ ID NO: 9, which encodes the amino acid sequence of SEQ ID NO: 10, which is a modified IL2 leader peptide. In certain embodiments, the nucleic acid sequence encoding a leader peptide operably linked to the trastuzumab light chain comprises the nucleic acid sequence of SEQ ID NO: 11.In certain embodiments, the nucleic acid sequence encoding a leader peptide operably linked to the trastuzumab light chain comprises the nucleic acid sequence of SEQ ID NO:11, which encodes the amino acid sequence of SEQ ID NO:12, a modified IL2 leader peptide.

[0037] In certain embodiments, the engineered nucleic acid sequence encoding a trastuzumab immunoglobulin comprises, from 5' to 3', an IL2 leader peptide, a trastuzumab heavy chain, optionally a furin cleavage sequence, a T2A linker, an IL2 leader peptide that is optionally a modified IL2 leader peptide, and a trastuzumab light chain. In certain embodiments, the engineered nucleic acid sequence encoding a trastuzumab immunoglobulin comprises, from 5' to 3', an IL2 leader peptide, a trastuzumab heavy chain, a furin cleavage sequence, a T2A linker, an IL2 leader peptide that is a modified IL2 leader peptide, and a trastuzumab light chain.

[0038] In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab heavy chain comprising the IL2 leader peptide comprises SEQ ID NO: 13. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab heavy chain comprising the IL2 leader peptide comprises a sequence at least 95% identical to SEQ ID NO: 13. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab heavy chain comprising the IL2 leader peptide comprises SEQ ID NO: 13, which encodes the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab light chain comprising the modified IL2 leader peptide comprises SEQ ID NO: 15. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab light chain comprising the modified IL2 leader peptide comprises a sequence at least 95% identical to SEQ ID NO: 15. In certain embodiments, the engineered nucleic acid sequence encoding the trastuzumab light chain comprising the modified IL2 leader peptide comprises SEQ ID NO: 15, which encodes the amino acid sequence of SEQ ID NO: 16.

[0039] In certain embodiments, an engineered nucleic acid sequence encoding a trastuzumab immunoglobulin comprising the nucleic acid sequence of SEQ ID NO: 17 comprises an IL2 leader peptide sequence, a trastuzumab heavy chain coding sequence, a T2A linker sequence, a modified IL2 leader peptide sequence, and a trastuzumab light chain coding sequence. In certain embodiments, an engineered nucleic acid sequence encoding a trastuzumab immunoglobulin comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 17 comprises an IL2 leader peptide sequence, a trastuzumab heavy chain coding sequence, a T2A linker sequence, a modified IL2 leader peptide sequence, and a trastuzumab light chain coding sequence. In certain embodiments, an engineered nucleic acid sequence encoding a trastuzumab immunoglobulin comprising the nucleic acid sequence of SEQ ID NO: 29 comprises an IL2 leader peptide sequence, a trastuzumab heavy chain coding sequence, a furin cleavage sequence, a T2A linker sequence, a modified IL2 leader peptide sequence, and a trastuzumab light chain coding sequence. In certain embodiments, an engineered nucleic acid sequence encoding a trastuzumab immunoglobulin comprising a nucleic acid sequence at least 95% identical to SEQ ID NO:29, the sequence comprises an IL2 leader peptide sequence, a trastuzumab heavy chain coding sequence, a furin cleavage sequence, a T2A linker sequence, a modified IL2 leader peptide sequence, and a trastuzumab light chain coding sequence.

[0040] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising: a nucleic acid sequence of SEQ ID NO: 7 encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO: 3 encoding a trastuzumab heavy chain; a nucleic acid sequence of SEQ ID NO: 31 encoding a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a nucleic acid sequence of SEQ ID NO: 32 encoding T2A; a nucleic acid sequence of SEQ ID NO: 7 encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO: 5 encoding a trastuzumab light chain.

[0041] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising: a nucleic acid sequence of SEQ ID NO:7 encoding the amino acid sequence of SEQ ID NO:8, which is a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3 encoding the amino acid sequence of SEQ ID NO:4, which is a trastuzumab heavy chain; a nucleic acid sequence of SEQ ID NO:31 encoding the amino acid sequence of SEQ ID NO:49, which is a furin cleavage site; a nucleic acid sequence encoding a "GSG" linker; and a linker comprising the nucleic acid sequence of SEQ ID NO:32 encoding the amino acid sequence of SEQ ID NO:51, which is T2A; a nucleic acid sequence of SEQ ID NO:7 encoding the amino acid sequence of SEQ ID NO:8, which is a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5 encoding the amino acid sequence of SEQ ID NO:6, which is a trastuzumab light chain.

[0042] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising: a nucleic acid sequence of SEQ ID NO: 7 encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO: 3 encoding a trastuzumab heavy chain; a nucleic acid sequence of SEQ ID NO: 31 encoding a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a linker comprising the nucleic acid sequence of SEQ ID NO: 32 encoding T2A; a nucleic acid sequence of SEQ ID NO: 9 encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO: 5 encoding a trastuzumab light chain.

[0043] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising: a nucleic acid sequence of SEQ ID NO:7 encoding the amino acid sequence of SEQ ID NO:8, which is a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3 encoding the amino acid sequence of SEQ ID NO:4, which is a trastuzumab heavy chain; a nucleic acid sequence of SEQ ID NO:31 encoding the amino acid sequence of SEQ ID NO:49, which is a furin cleavage site; a nucleic acid sequence encoding a "GSG" linker; and a linker comprising a nucleic acid sequence of SEQ ID NO:32 encoding the amino acid sequence of SEQ ID NO:51, which is T2A; a nucleic acid sequence of SEQ ID NO:9 encoding the amino acid sequence of SEQ ID NO:10, which is a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5 encoding the amino acid sequence of SEQ ID NO:6, which is a trastuzumab light chain.

[0044] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising: a nucleic acid sequence of SEQ ID NO:7 encoding a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain; a nucleic acid sequence of SEQ ID NO:31 encoding a furin cleavage site, a nucleic acid sequence encoding a "GSG" linker, and a nucleic acid sequence of SEQ ID NO:32 encoding a T2A linker; a nucleic acid sequence of SEQ ID NO:11 encoding a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain.

[0045] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising: a nucleic acid sequence of SEQ ID NO:7 encoding the amino acid sequence of SEQ ID NO:8, which is a leader peptide operably linked to a trastuzumab heavy chain; a nucleic acid sequence comprising SEQ ID NO:3 encoding the amino acid sequence of SEQ ID NO:4, which is a trastuzumab heavy chain; a nucleic acid sequence of SEQ ID NO:31 encoding the amino acid sequence of SEQ ID NO:49, which is a furin cleavage site; a nucleic acid sequence encoding a "GSG" linker; and a linker comprising a nucleic acid sequence of SEQ ID NO:32 encoding the amino acid sequence of SEQ ID NO:51, which is T2A; a nucleic acid sequence of SEQ ID NO:11 encoding the amino acid sequence of SEQ ID NO:12, which is a leader peptide operably linked to a trastuzumab light chain; and a nucleic acid sequence comprising SEQ ID NO:5 encoding the amino acid sequence of SEQ ID NO:6, which is a trastuzumab light chain.

[0046] A "nucleic acid" as described herein can be RNA, DNA, or modifications thereof, can be single-stranded or double-stranded, and can be selected from the group including, for example, a nucleic acid encoding a protein of interest, an oligonucleotide, a nucleic acid analog, such as peptide nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), locked nucleic acid (LNA), and the like. Nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotide (e.g., peptide nucleic acid oligomers). Those skilled in the art will understand that functional variants of these nucleic acid molecules are also intended to be part of the present invention. Functional variants are nucleic acid sequences that can be directly translated using the standard genetic code to provide the same amino acid sequence as that translated from the parent nucleic acid molecule. Such nucleic acid sequences include, for example, but are not limited to, In certain embodiments, the miRNA is a dorsal root ganglion (drg)-specific miRNA target sequence. In certain embodiments, the nucleic acid sequence further comprises at least one, at least two, at least three, or preferably at least four tandem repeats of a dorsal root ganglion (drg)-specific miRNA target sequence. In certain embodiments, the nucleic acid sequence further comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or preferably at least eight tandem repeats of a dorsal root ganglion (drg)-specific miRNA target sequence. See, e.g., PCT / US19 / 67872, filed December 20, 2019, now published as WO2020 / 132455, which is incorporated herein by reference. See also, International Patent Application No. PCT / US21 / 32003, filed May 12, 2021, and now published WO2021 / 231579A1, which are incorporated herein by reference. See also, U.S. Provisional Patent Application No. 63 / 279,561, filed November 15, 2021, which is incorporated herein by reference in its entirety.

[0047] In certain embodiments, recombinant nucleic acid molecules and other constructs encoding Trastuzuma immunoglobulins encompassed by the present invention and useful for generating expression cassettes and vector genomes can be engineered for expression in mammalian cells, such as yeast cells, insect cells, or human cells. Methods are known and have been previously described (e.g., WO96 / 09378). A sequence is considered engineered if at least one non-preferred codon compared to the wild-type (WT) sequence is replaced by a more preferred codon. As used herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon that codes for the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The codon usage frequency of a particular organism can be found in a codon frequency table, for example, at kazusa.jp / codon. Preferably, more than one non-preferred codon, preferably most or all non-preferred codons, are replaced by a more preferred codon. Preferably, the codon most frequently used in the organism is used in the engineered sequence. Substitution with preferred codons generally results in higher expression. It will also be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, many different nucleic acid molecules can code for the same polypeptide. It will also be understood that those skilled in the art may use routine techniques to make nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acid molecule to reflect the codon usage of any particular host organism in which the polypeptide is expressed. Thus, unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. The nucleic acid sequence can be cloned using routine molecular biology techniques or generated de novo by DNA synthesis, which can be carried out using routine procedures by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins).

[0048] By "engineered" it is meant that the nucleic acid sequences encoding the Trastuzuma immunoglobulins described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which introduces the Trastuzuma immunoglobulins carried therein into a host cell, e.g., to generate a non-viral delivery system (e.g., RNA-based systems, naked DNA, etc.), or to generate a viral vector in a packaging host cell, and / or for delivery to a host cell of interest. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. Methods used to make such engineered constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0049] The terms "percent identity (%)", "sequence identity", "percent sequence identity", or "percent identical" in the context of nucleic acid sequences refer to residues in two sequences that are the same when aligned in correspondence, and refer to subsequences that are the same over a particular region (e.g., any one of the modified ORFs provided herein when compared and aligned for maximum correspondence over a comparison window or designated region), or have a particular percentage of identical amino acid residues or nucleotides (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (e.g., at the NCBI website, etc.). The length of sequence identity comparison may be, and preferably is, over the entire length of the genome, the entire length of the gene coding sequence, or a fragment of at least about 500-5000 nucleotides, however identity between smaller fragments, for example, at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 or more nucleotides, may also be desired.

[0050] Percent identity can be readily determined for amino acid sequences spanning the full length polypeptide, polypeptide, about 32 amino acids, about 330 amino acids, or peptide fragments thereof, or the corresponding nucleic acid sequence encoding the sequence. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 700. In general, when referring to "identity", "homology", or "similarity" between two different sequences, the "identity", "homology", or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence.

[0051] The alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, including, for example, "Clustal X", "Clustal Omega", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of these programs are used with default settings, but those skilled in the art may change these settings as necessary. Alternatively, those skilled in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithm and program. See, for example, JD Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0052] Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal W", "Clustal Omega", "CAP Sequence Assembly", "BLAST", "MAP" and "MEME" and are accessible through web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility is also used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ using its default parameters (word size 6 and NOPAM factor for scoring matrix) provided in GCG version 6.1, which is incorporated herein by reference.

[0053] It is to be understood that the compositions in Trastuzuma immunoglobulin and Trastuzuma immunoglobulin coding sequences described herein are intended to apply to other compositions, regimens, aspects, embodiments, and methods described throughout the specification.

[0054] Expression cassette Provided herein is a gene therapy vector comprising an expression cassette comprising an engineered nucleic acid sequence comprising a coding sequence for an anti-Her2 antibody, i.e., a trastuzumab immunoglobulin, including heavy and light chains, operably linked to a regulatory sequence directing its expression. Also provided herein is a recombinant nucleic acid molecule comprising an expression cassette comprising a nucleic acid sequence encoding a trastuzumab immunoglobulin as described herein. Also provided herein is a recombinant nucleic acid molecule comprising a vector genome comprising a nucleic acid sequence encoding a trastuzumab immunoglobulin as described herein.

[0055] As used herein, an "expression cassette" refers to a nucleic acid molecule that includes a biologically useful nucleic acid sequence and a regulatory sequence operably linked thereto that directs or regulates the transcription, translation, and / or expression of the nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme, or other useful gene product, mRNA, etc.) and the gene product. As used herein, an "operably linked" sequence includes both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in cis or trans with the nucleic acid sequence. Such regulatory sequences typically include, for example, one or more of a promoter, enhancer, intron, Kozak sequence, polyadenylation sequence, and TATA signal. An expression cassette may include, among other elements, regulatory sequences upstream (5') of the gene sequence, e.g., one or more of a promoter, enhancer, intron, etc., and enhancer, or regulatory sequences downstream (3') of the gene sequence, e.g., one or more of a 3' untranslated region (3'UTR) that includes a polyadenylation site. In certain embodiments, the regulatory sequence is operably linked to the nucleic acid sequence of the gene product, and the regulatory sequence is separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., the 5' untranslated region (5'UTR). In certain embodiments, the expression cassette comprises the nucleic acid sequence of one or more gene products. In some embodiments, the expression cassette can be a monocistronic or bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell. Typically, such expression cassettes for producing viral vectors comprise coding sequences of gene products described herein adjacent to packaging signals of the viral genome, and other expression control sequences such as those described herein. In certain embodiments, the vector genome can comprise two or more expression cassettes.

[0056] The term "exogenous" as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the chromosome or at the location present in the host cell. An exogenous nucleic acid sequence also refers to a sequence that is derived from and inserted into the same host cell or subject, but exists in a non-native state, e.g., in a different copy number or under the control of different regulatory elements.

[0057] The term "heterologous" when used to describe a nucleic acid sequence or protein means that the nucleic acid or protein is derived from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. The term "heterologous" when used in reference to a protein, or a nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or nucleic acid is present in another sequence or subsequence that is not found in the same relationship to each other in nature.

[0058] In certain embodiments, the regulatory sequence comprises a promoter, hi certain embodiments, the regulatory sequence comprises one or more introns, one or more enhancers, and a polyadenylation (polyA) signal sequence.

[0059] In one embodiment, the regulatory sequence comprises a promoter. In one embodiment, the promoter is a chicken β-actin (also referred to as chicken beta-actin, CB, or CBA) promoter. Various chicken beta-actin promoters have been reported alone or in combination with various enhancer elements (e.g., CB7 is a chicken beta-actin promoter with a cytomegalovirus enhancer element, which is a CAG promoter, which contains the promoter, the first exon and first intron of chicken beta actin, and the splice acceptor of the rabbit beta globin gene), the CBh promoter [SJ Gray et al, Hu Gene Ther, 2011 Sep;22(9):143-1 153]. In a particular embodiment, the promoter is a CMV promoter. In a particular embodiment, the CB promoter comprises the nucleic acid sequence of SEQ ID NO:20.

[0060] In further embodiments, the promoter is a CB7 (also called hybrid CB7) promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer and a chicken beta-actin promoter, optionally with a spacer sequence, optionally comprising a chicken beta actin intron, and further comprising a chicken beta-actin splicing donor (an exon sequence, comprising a chicken beta actin intron) and a rabbit beta globin splicing acceptor. In certain embodiments, the CMV IE promoter comprises the nucleic acid sequence of SEQ ID NO: 19. In certain embodiments, the CB7 promoter comprises the nucleic acid sequence of SEQ ID NO: 21. See, for example, the cytomegalovirus (CMV) immediate early enhancer (260 bp, C4, GenBank No. K03104.1). The chicken beta-actin promoter (281 bp, CB, GenBank No. X00182.1). In certain embodiments, the promoter is a ubiquitin C (UbC) promoter. See also WO2001 / 091800. See, e.g., GenBank Accession Nos. AF232305 (rat) and D63791 (human), respectively. In certain embodiments, the UbC promoter comprises the nucleic acid sequence of SEQ ID NO: 24. Still other promoters and / or enhancers may be selected.In another embodiment, suitable promoters include, but are not limited to, the elongation factor 1 alpha (EF1 alpha) promoter (see, e.g., Kim DW et al, Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23), the synapsin 1 promoter (see, e.g., Kuegler S et al, Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47), the neuron-specific enolase (NSE) promoter (see, e.g., Kim J et al, Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology. 2004 Feb;145(2):613-9. Epub 2003 Oct 16), or the CB6 promoter (see, e.g., Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene, Mol Biotechnol. 2016 Jan;58(1):30-6. doi:10.1007 / s12033-015-9899-5). In yet other embodiments, multiple enhancers and / or promoters may be included.

[0061] In certain embodiments, additional or alternative promoter sequences may be included as part of the expression control sequence (regulatory sequence), for example, located between the selected 5'ITR sequence and the coding sequence. Constitutive promoters, regulatable promoters [see, e.g., WO2011 / 126808 and WO2013 / 04943], tissue-specific promoters, or promoters responsive to physiological cues may be utilized in the vectors described herein. The promoter(s) may be selected from different sources, for example, human cytomegalovirus immediate early (CMV IE) enhancer / promoter, SV40 early enhancer / promoter, JC polymovirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency-associated promoter (LAP), Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), and chicken beta-actin promoter.

[0062] In one embodiment, the expression cassette is designed for expression and secretion in a human subject. In one embodiment, the expression cassette is designed for expression in the central nervous system (CNS), including the cerebrospinal fluid and the brain. In a further embodiment, the expression cassette is useful for expression in both the CNS and the whole body. A suitable promoter can be selected, including, but not limited to, a constitutive promoter, a tissue-specific promoter, or an inducible / regulatable promoter. An example of a constitutive promoter is the chicken beta-actin promoter. See also CB7, above. Examples of promoters that are tissue specific include those from the liver (albumin, Miyatake et al., (1997) J. Virol., 71:5124-32; hepatitis B virus core promoter, Sandig et al., (1996) Gene Ther., 3:1002-9; alpha-fetoprotein (AFP): Arbuthnot et al., (1996) Hum. Gene Ther., 7:1503 14), neurons (neuron-specific enolase (NSE) promoter, Andersen et al., (1993) Cell. Mol. Neurobiol., 13:503 15; neurofilament light chain gene, Piccioli et al., (1991) Proc. Natl. Acad. Sci. USA, 88:5611 5; and the neuron-specific vgf gene: Piccioli et al. al., (1995) Neuron, 15:373-84), as well as other tissues. Alternatively, regulatable promoters may be selected. See, for example, WO2011 / 126808B2, which is incorporated herein by reference.

[0063] In addition to a promoter, the vector may include one or more other suitable transcription initiation sequences, transcription termination sequences, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA (e.g., WPRE), sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, optionally, sequences that enhance secretion of the encoded product.

[0064] In one embodiment, the regulatory sequence further comprises an enhancer. In one embodiment, the regulatory sequence comprises one enhancer. In another embodiment, the regulatory sequence comprises two or more expression enhancers. These enhancers can be the same or different. For example, the enhancer may comprise an alpha mic / bik enhancer, or a CMV IE enhancer. This enhancer may be present in two copies located adjacent to each other. Alternatively, the double copies of the enhancer are separated by one or more sequences.

[0065] In one embodiment, the regulatory sequence further comprises an intron. In a further embodiment, the intron is a chicken beta-actin intron. In one embodiment, the intron is 875 bp (GenBank No. X00182.1). In one embodiment, the intron is a 973 bp intron from the chicken beta-actin gene (GenBank No. X00182.1). In a particular embodiment, the chicken beta-actin intron comprises the nucleic acid sequence of SEQ ID NO: 34. In a particular embodiment, the intron is a chimeric intron (CI)-a hybrid intron consisting of a human beta-globin splice donor and an immunoglobulin G (IgG) splice acceptor element. In a particular embodiment, the intron is a chimeric intron comprising a chicken beta-actin splicing donor (including exon sequences), a chicken beta-actin intron, and a rabbit beta-globin splicing acceptor. In certain embodiments, the intron is a chimeric intron comprising a chicken beta actin intron comprising the nucleic acid sequence of SEQ ID NO: 51. In another embodiment, a chimeric intron available from Promega® is used. In certain embodiments, the chimeric intron is a Promega® intron comprising the nucleic acid sequence of SEQ ID NO: 22. Other suitable introns include those known in the art, may be a human β-globulin intron, and / or commercially available introns, and those described in WO2011 / 126808.

[0066] In one embodiment, the regulatory sequence further comprises a polyadenylation signal (polyA). Examples of suitable polyA sequences include, for example, rabbit globin (RBG or rBG) polyA, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Optionally, one or more sequences may be selected to stabilize the mRNA. In a particular embodiment, the polyA is rabbit beta globin polyA (rabbit globin polyA or rBG). See, for example, WO2014 / 151341. In a particular embodiment, a human growth hormone (hGH) polyadenylation sequence, SV40 polyA, or synthetic polyA may be included in the expression cassette. In a particular embodiment, SV40 polyA is selected. In a particular embodiment, the SV40 polyA comprises the nucleic acid sequence of SEQ ID NO:23.

[0067] In certain embodiments, the expression cassette comprises a trastuzumab coding sequence, and may include other regulatory sequences therefor, such that the necessary regulatory sequences are operably linked to the trastuzumab coding sequence in a manner that allows for its transcription, translation, and / or expression in the target cell.

[0068] In certain embodiments, the target cell can be a central nervous system cell. In certain embodiments, the target cell is one or more of an excitatory neuron, an inhibitory neuron, a glial cell, a cortical cell, a frontal cortex cell, a cerebral cortex cell, and a spinal cord cell. In certain embodiments, the target cell is in the leptomeninges (LM) of the CNS. In certain embodiments, the target cell is in the parenchyma of the CNS.

[0069] In certain embodiments, the expression cassette comprises: (i) a promoter comprising an enhancer and a promoter, optionally having a spacer sequence therebetween; (ii) an intron; (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain; (v) optionally a furin cleavage site; (vi) a T2A element linker; (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; (viii) a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain; and (ix) an SV40 polyadenylation (polyA) sequence.

[0070] In certain embodiments, the expression cassette comprises (i) a promoter comprising an enhancer and a promoter, optionally having a spacer sequence therebetween, (ii) an intron, (iii) a Kozak sequence, (iv) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (v) a nucleic acid sequence comprising SEQ ID NO: 3 encoding a trastuzumab heavy chain, (vi) a furin cleavage site, (vii) a T2A element linker, (viii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (ix) a nucleic acid sequence comprising SEQ ID NO: 5 encoding a trastuzumab light chain, and (x) an SV40 polyadenylation (polyA) sequence.

[0071] In certain embodiments, the expression cassette comprises: (i) a promoter element comprising a chicken beta actin promoter (CB) and enhancer; (ii) an intron, which is a chicken beta actin intron (CI); (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO: 3 encoding a trastuzumab heavy chain; (v) a T2A element linker; (vi) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; (vii) a nucleic acid sequence comprising SEQ ID NO: 5 encoding a trastuzumab light chain; and (viii) an SV40 polyadenylation (polyA) sequence.

[0072] In certain embodiments, the expression cassette comprises: (i) a promoter element comprising a chicken beta-actin promoter (CB) and enhancer; (ii) an intron, which is a chicken beta-actin intron (CI); (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3, or a sequence at least 95% identical to SEQ ID NO:3, encoding a trastuzumab heavy chain; (v) a furin cleavage site; (vi) a T2A element linker; (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; (viii) a nucleic acid sequence comprising SEQ ID NO:5, or a sequence at least 95% identical to SEQ ID NO:5, encoding a trastuzumab light chain; and (ix) an SV40 polyadenylation (polyA) sequence.

[0073] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter comprising a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron comprising a chicken beta actin intron (CI); (ii) a Kozak sequence; (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3, or a sequence at least 95% identical to SEQ ID NO:3, encoding a trastuzumab heavy chain; (v) optionally a furin cleavage site; (vi) a T2A element linker; (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; (viii) a nucleic acid sequence comprising SEQ ID NO:5, or a sequence at least 95% identical to SEQ ID NO:5, encoding a trastuzumab light chain; and (ix) an SV40 polyadenylation (polyA) sequence.

[0074] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter comprising a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron comprising a chicken beta-actin splicing donor (including exon sequences), a chicken beta actin intron, and a rabbit b-globin splicing acceptor; (ii) a Kozak sequence; (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain, or a sequence at least 95% identical to SEQ ID NO:3; (v) a furin cleavage site; (vi) a T2A element linker; (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; (viii) a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain, or a sequence at least 95% identical to SEQ ID NO:5; and (ix) an SV40 polyadenylation (polyA) sequence.

[0075] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter comprising the nucleic acid sequence of SEQ ID NO:21; (ii) a Kozak sequence; (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide comprising the nucleic acid sequence of SEQ ID NO:7 operably linked to a trastuzumab heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain, or a sequence at least 95% identical to SEQ ID NO:3; (v) a furin cleavage site comprising the nucleic acid sequence of SEQ ID NO:31; (vi) a T2A element linker comprising the nucleic acid sequence of SEQ ID NO:32; (vii) a nucleic acid sequence encoding a leader peptide comprising the nucleic acid sequence of SEQ ID NO:9 operably linked to a trastuzumab light chain; (viii) a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain, or a sequence at least 95% identical to SEQ ID NO:5; and (ix) an SV40 polyadenylation (polyA) sequence comprising the nucleic acid sequence of SEQ ID NO:23.

[0076] In certain embodiments, the expression cassette comprises: (i) a promoter that is a CB7 hybrid promoter comprising the nucleic acid sequence of SEQ ID NO:21; (ii) a Kozak sequence; (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide comprising the nucleic acid sequence of SEQ ID NO:7 operably linked to a trastuzumab heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain, or a sequence at least 95% identical to SEQ ID NO:3; (v) a furin cleavage site followed by a T2A element linker connected via a "GSG" linker comprising the nucleic acid sequence of SEQ ID NO:33 (furin / T2A); (vi) a nucleic acid sequence encoding a leader peptide comprising the nucleic acid sequence of SEQ ID NO:9 operably linked to a trastuzumab light chain; (vii) a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain, or a sequence at least 95% identical to SEQ ID NO:5; and (viii) an SV40 polyadenylation (polyA) sequence comprising the nucleic acid sequence of SEQ ID NO:23.

[0077] In certain embodiments, the expression cassette comprises (i) a promoter that is a CB7 hybrid promoter comprising a CMV IE enhancer and a chicken beta-actin promoter, and a chimeric intron comprising a chicken beta actin intron (CI); (ii) a Kozak sequence; (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain, or a sequence at least 95% identical to SEQ ID NO:3; (v) a furin cleavage site; (vi) a T2A element linker; (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; (viii) a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain, or a sequence at least 95% identical to SEQ ID NO:5; and (ix) an SV40 polyadenylation (polyA) sequence.

[0078] In certain embodiments, the expression cassette comprises: (i) a promoter that is a Ubiquitin C (UbC) promoter; (ii) an intron that is a chimeric intron comprising a Promega intron (PI); (iii) a Kozak sequence; (iv) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; (v) a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain, or a sequence at least 95% identical to SEQ ID NO:3; (vi) optionally a Furin cleavage site; (vii) a T2A element linker; (viii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; (ix) a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain, or a sequence at least 95% identical to SEQ ID NO:5; and (x) an SV40 polyadenylation (polyA) sequence.

[0079] In certain embodiments, the expression cassette comprises: (i) a promoter that is a Ubiquitin C (UbC) promoter; (ii) an intron that is a chimeric intron comprising a Promega intron (PI); (iii) a Kozak sequence; (iv) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain; (v) a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain, or a sequence at least 95% identical to SEQ ID NO:3; (vi) a Furin cleavage site; (vii) a T2A element linker; (viii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain; (ix) a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain, or a sequence at least 95% identical to SEQ ID NO:5; and (x) an SV40 polyadenylation (polyA) sequence.

[0080] In certain embodiments, the expression cassette comprises: (i) a promoter (SEQ ID NO:24) that is a Ubiquitin C (UbC) promoter; (ii) an intron (SEQ ID NO:22) that is a chimeric intron comprising a Promega intron (PI); (iii) a Kozak sequence; (iv) at least one ORF comprising a nucleic acid sequence (SEQ ID NO:7) encoding a leader peptide operably linked to a trastuzumab heavy chain; (v) a nucleic acid sequence comprising SEQ ID NO:3 encoding a trastuzumab heavy chain, or a sequence at least 95% identical to SEQ ID NO:3; (vi) a Furin cleavage site (SEQ ID NO:31); (vii) a T2A element linker (SEQ ID NO:32); (viii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain (SEQ ID NO:11); (ix) a nucleic acid sequence comprising SEQ ID NO:5 encoding a trastuzumab light chain, or a sequence at least 95% identical to SEQ ID NO:5; and (x) an SV40 polyadenylation (polyA) sequence (SEQ ID NO:23).

[0081] In certain embodiments, the expression cassette comprises (i) a promoter that is a ubiquitin C (UbC) promoter (SEQ ID NO: 24); (ii) an intron that is a chimeric intron containing a Promega intron (PI) (SEQ ID NO: 22); (iii) a Kozak sequence; (iv) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain (SEQ ID NO: 7); and (v) a nucleic acid sequence encoding a trastuzumab heavy chain, comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, and a nucleic acid sequence encoding a leader peptide operably linked to ... and a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, comprising a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, and a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain. (vi) a furin cleavage site (furin / T2A) connected to a T2A element linker via a "GSG" linker (SEQ ID NO:33); (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain (SEQ ID NO:11); (viii) a nucleic acid sequence encoding a trastuzumab light chain comprising SEQ ID NO:5, or a sequence at least 95% identical to SEQ ID NO:5; and (ix) an SV40 polyadenylation (polyA) sequence (SEQ ID NO:23).

[0082] In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO:2 (CB.CI.IL2.V1.trastuzumab-coGW.SV40), or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO:2 (CB.CI.IL2.V1.trastuzumab-coGW.SV40), or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO:26 (UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40), or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 26 (UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40), or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 28 (CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40), or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 28 (CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40), or a sequence at least 99% identical thereto.

[0083] It is to be understood that the compositions in the expression cassettes described herein are intended to apply to other compositions, regimens, aspects, embodiments and methods described throughout the specification.

[0084] Recombinant adeno-associated virus Provided herein is a recombinant adeno-associated virus (rAAV) comprising an engineered nucleic acid sequence encoding trastuzumab immunoglobulin (e.g., rAAV.trastuzumab-coGW). Provided herein is a recombinant adeno-associated virus (rAAV) useful for treating metastatic HER2-positive cancer in the brain. The rAAV comprises (a) an AAV capsid, and (b) a vector genome packaged in the AAV capsid of (a). Suitably, the selected AAV capsid targets the cells to be treated. In certain embodiments, the capsid is from clade F. However, in certain embodiments, another AAV capsid source may be selected, i.e., clade A. In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is an AAVrh91 capsid. In certain embodiments, the AAV capsid is an AAVhu95 capsid. In certain embodiments, the AAV capsid is an AAVhu96 capsid. The vector genome comprises an AAV 5' inverted terminal repeat (ITR), at least one open reading frame (ORF) comprising a trastuzumab heavy chain and a trastuzumab light chain, and nucleic acid sequences operably linked thereto that regulate expression of the trastuzumab heavy chain and light chain, and an AAV 3' ITR.

[0085] As used herein, the term "vector genome" refers to a nucleic acid molecule that can be packaged into a viral capsid (e.g., an AAV capsid) and delivered to a host cell or a patient's cell. In certain embodiments, the vector genome comprises terminal repeats (e.g., AAV inverted terminal repeats (ITRs) at the extreme 5' and 3' ends necessary for packaging the vector genome into a capsid, and between them an expression cassette that comprises a trastuzumab immunoglobulin gene (e.g., trastuzumab-coGW) described herein operably linked to sequences that direct its expression.

[0086] The AAV sequence of the vector typically includes cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are approximately 145 base pairs (bp) in length. Preferably, substantially the entire sequence encoding the ITRs is used in the molecule, although some minor modification of these sequences is tolerated. The ability to modify these ITR sequences is within the skill of 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)). One example of such a molecule used in the present invention is a "cis-acting" plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are from a different AAV than the one supplying the capsid. In one embodiment, the ITR sequences are from AAV2. A shortened version of the 5'ITR, termed ΔITR, has been described, in which the D sequence and terminal separation sites (trs) are deleted. In certain embodiments, the vector genome (e.g., of a plasmid) contains a shortened AAV2 ITR of 130 base pairs, in which the external A element is deleted. The shortened ITR can be restored to the wild-type length of 145 base pairs during vector DNA amplification using the internal A element as a template and packaging into capsids that form viral particles. In other embodiments, full-length AAV 5' and 3' ITRs are used. However, ITRs from other AAV sources can be selected. When the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped, however, other configurations of these elements may be suitable.

[0087] In one embodiment, the rAAV.trastuzumab-coGW vector comprises an AAV capsid and, packaged therein, a vector genome that comprises at least one element heterologous to the AAV capsid. In one embodiment, the vector genome comprises, from 5' to 3': (a) an AAV 5'ITR, (b) optionally an enhancer, (c) a promoter, (d) an intron, (e) a leader sequence and a trastuzumab heavy chain coding sequence, (f) optionally a furin cleavage sequence, (g) a T2A linker, (h) a leader sequence and a trastuzumab light chain coding sequence, (i) a polyA signal, and (j) an AAV 3'ITR.

[0088] In one embodiment, the vector genome comprises, from 5' to 3': (a) the AAV 5'ITR; (b) a CB7 promoter comprising a CMV IE enhancer, a chicken beta actin promoter and a chimeric intron comprising a chicken beta-actin splicing donor (including exon sequences), a chicken beta actin intron, and a rabbit b-globin splicing acceptor, optionally with a spacer sequence; (c) a Kozak sequence; (d) a leader sequence and a trastuzumab heavy chain coding sequence; (e) a furin cleavage sequence; (f) a T2A linker; (g) a leader sequence and a trastuzumab light chain coding sequence; (h) an SV40 polyA signal; and (i) an AAV 3'ITR. In one embodiment, the vector genome comprises, from 5' to 3': (a) the AAV 5'ITR; (b) a CB7 promoter comprising a CMV IE enhancer, a chicken beta actin promoter and a chimeric intron comprising a chicken beta-actin splicing donor (including exon sequences), a chicken beta actin intron, and a rabbit b-globin splicing acceptor, optionally with a spacer sequence; (c) a Kozak sequence; (d) a leader sequence and a trastuzumab heavy chain coding sequence; (e) a T2A linker; (f) a leader sequence and a trastuzumab light chain coding sequence; (g) an SV40 polyA signal; and (h) an AAV 3'ITR.

[0089] In one embodiment, the vector genome comprises, from 5' to 3': (a) AAV 5'ITR, (b) chicken beta actin promoter, (c) chicken beta actin intron (CI), (d) leader sequence and trastuzumab heavy chain coding sequence, (e) furin cleavage sequence, (f) T2A linker, (g) leader sequence and trastuzumab light chain coding sequence, (h) SV40 polyA signal, and (i) AAV3'ITR. In one embodiment, the vector genome comprises, from 5' to 3': (a) AAV 5'ITR, (b) chicken beta actin promoter, (c) chicken beta actin intron (CI), (d) leader sequence and trastuzumab heavy chain coding sequence, (e) T2A linker, (f) leader sequence and trastuzumab light chain coding sequence, (g) SV40 polyA signal, and (h) AAV3'ITR.

[0090] In one embodiment, the vector genome comprises, from 5' to 3': (a) an AAV 5' ITR, (b) a ubiquitin C (UbC) promoter, (c) a chimeric (Promega) intron (PI), (d) a Kozak sequence, (e) a leader sequence and a trastuzumab heavy chain coding sequence, (f) a furin cleavage sequence, (g) a T2A linker, (h) a leader sequence and a trastuzumab light chain coding sequence, (i) an SV40 polyA signal, and (j) an AAV 3' ITR. In one embodiment, the vector genome comprises, from 5' to 3': (a) the AAV 5' ITR, (b) a ubiquitin C (UbC) promoter, (c) a chimeric (Promega) intron (PI), (d) a Kozak sequence, (e) a leader sequence and a trastuzumab heavy chain coding sequence, (f) a T2A linker, (g) a leader sequence and a trastuzumab light chain coding sequence, (h) an SV40 polyA signal, and (i) an AAV 3' ITR.

[0091] In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 1 (5'-ITR.CB.CI.IL2.V1.trastuzumab-coGW.SV40.3'-ITR), or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 1 (5'-ITR.CB.CI.IL2.V1.trastuzumab-coGW.SV40.3'-ITR), or a sequence at least 99% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 25 (5'-ITR.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR), or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 25 (5'-ITR.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR), or a sequence at least 99% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 27 (5'-ITR.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR), or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 27 (5'-ITR.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR), or a sequence at least 99% identical thereto.

[0092] In certain embodiments, the AAV capsid is a clade F AAV capsid, and a clade F AAV capsid can be an AAVhu68 capsid (see, e.g., US2020 / 0056159, PCT / US21 / 55436, SEQ ID NOs: 37 and 38 for nucleic acid sequences, and SEQ ID NO: 39 for amino acid sequence), an AAVhu95 capsid (see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 201, and International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022, SEQ ID NOs: 43 and 44 (hu95 nucleic acid sequence)). and SEQ ID NO:45 (hu95 amino acid sequence), or AAVhu96 capsid (see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 201, and International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022, SEQ ID NOs:46 and 47 (hu96 nucleic acid sequence) and SEQ ID NO:48 (hu96 amino acid sequence)), AAV9 capsid (SEQ ID NO:54 for the nucleic acid sequence and SEQ ID NO:55 for the amino acid sequence). In certain embodiments, the AAV capsid is a clade A capsid, such as an AAVrh91 capsid (nucleic acid sequences of SEQ ID NOs:40 and 41, amino acid sequence of SEQ ID NO:42). See PCT / US20 / 030266, filed April 29, 2020, now published WO2020 / 223231 (incorporated by reference into this specification), and International Application No. PCT / US21 / 45945, filed August 13, 2021 (incorporated by reference into this specification).

[0093] In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is an AAV9 capsid. In certain embodiments, the AAV capsid is an AAVhu95 capsid. In certain embodiments, the AAV capsid is an AAVhu96 capsid.

[0094] In certain embodiments, the AAV capsid for the compositions and methods described herein is selected based on the target cell. In certain embodiments, the AAV capsid transduces CNS cells and / or PNS cells. In certain embodiments, other AAV capsids can be selected. The AAV capsid is selected from cy02 capsid, rh43 capsid, AAV8 capsid, rh01 capsid, AAV9 capsid, rh8 capsid, rh10 capsid, bb01 capsid, hu37 capsid, rh02 capsid, rh20 capsid, rh39 capsid, rh64 capsid, AAV6 capsid, AAV1 capsid, hu44 capsid, hu48 capsid, cy05 capsid, hu11 capsid, hu32 capsid, pi2 capsid, or variations thereof. In certain embodiments, the AAV capsid is a clade F capsid, such as an AAV9 capsid, an AAVhu68 capsid, a hu31 capsid, a hu32 capsid, or a variation thereof. See, for example, WO2005 / 033321, published April 14, 2015, WO2018 / 160582, and US2015 / 0079038, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV capsid is a non-clade F capsid, such as a clade A, B, C, D, or E capsid. In certain embodiments, the non-clade F capsid is AAV1 or a variation thereof. In certain embodiments, the AAV capsid transduces a target cell other than a nervous system cell. In certain embodiments, the AAV capsid is a clade A capsid (e.g., AAV1, AAV6, AAVrh91), a clade B capsid (e.g., AAV2), a clade C capsid (e.g., hu53), a clade D capsid (e.g., AAV7), or a clade E capsid (e.g., rh10).

[0095] The AAV capsid is an assembly of a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 proteins, and a heterogeneous population of vp3 proteins. As used herein, when used to refer to vp capsid proteins, the term "heterogeneous" or any grammatical variation thereof refers to a population of non-identical members, for example, having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences.

[0096] As used herein, the term "heterologous" or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a population of non-identical members, e.g., having vp1, vp2, or vp3 (also referred to as VP1, VP2, VP3, or Vp1, Vp2, Vp3) monomers (proteins) with different modified amino acid sequences. The term "heterologous population" used in reference to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of vp1, vp2, and vp3 proteins within a capsid. AAV capsids contain subpopulations within vp1, vp2, and vp3 proteins with predicted amino acid residue modifications. These subpopulations contain, at a minimum, specific deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, where deamidation results in an amino acid change and other optional modifications.

[0097] In certain embodiments, AAV capsids are provided that have a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3) that contain multiple highly deamidated "NG" positions. In certain embodiments, the highly deamidated positions are at the positions shown below with reference to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified such that the referenced "NG" is removed and a mutated "NG" is engineered into another position.

[0098] As used herein, the terms "target cell" and "target tissue" may refer to any cell or tissue intended to be transduced by the subject AAV vector. The terms may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (visual cells), or heart.

[0099] Additionally, the present specification provides an expression cassette comprising: (a) an AAV 5' inverted terminal repeat (ITR); (b) at least one open reading frame (ORF) comprising a trastuzumab heavy chain and a trastuzumab light chain, and a nucleic acid sequence operably linked thereto that regulates expression of the trastuzumab heavy chain and the trastuzumab light chain; and (c) an AAV and a 3'ITR, wherein the expression cassette comprises at least one ORF comprising (i) a promoter that is a ubiquitin C (UbC) promoter, and (ii) an intron that is a chimeric intron (e.g., available from Promega), and (iii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO: 3 encoding a trastuzumab heavy chain, (v) a furin cleavage site, (vi) a T2A linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to a trastuzumab light chain, (viii) a nucleic acid sequence comprising SEQ ID NO: 5 encoding a trastuzumab light chain, (ix) an SV40 polyadenylation (polyA) sequence, and the expression cassette further comprises a spacer sequence. In certain embodiments, the recombinant nucleic acid molecule comprises an expression cassette comprising the nucleic acid sequence of SEQ ID NO: 26. In certain embodiments, the recombinant nucleic acid molecule comprises a vector genome comprising the nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, the recombinant nucleic acid molecule is for use in the rAAV production system described herein.

[0100] Additionally, provided herein is a rAAV production system useful for producing the rAAV described herein. The production system comprises a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein, (b) a vector genome, and (c) sufficient AAV rep and helper functions to permit packaging of the vector genome into an AAV capsid. In certain embodiments, the vector genome is SEQ ID NO:1. In certain embodiments, the vector genome is SEQ ID NO:25. In certain embodiments, the vector genome is SEQ ID NO:27. In certain embodiments, the cell culture is a bacterial cell culture. In certain embodiments, the cell culture is a mammalian cell culture. In certain embodiments, the cell culture is a human embryonic kidney 293 (HEK293) cell culture. In certain embodiments, the cell culture is a suspension cell culture. In certain embodiments, the AAV rep is derived from a different AAV. In certain embodiments, the AAV rep is derived from AAV2. In certain embodiments, the AAV rep coding sequence and cap gene are on the same nucleic acid molecule, optionally with a spacer between the rep sequence and the cap gene.

[0101] For use in producing AAV viral vectors (e.g., recombinant (r)AAV), the vector genome can be carried on any suitable vector, e.g., a plasmid, that is delivered to a packaging host cell. Plasmids useful in the present invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic, insect, and mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by one of skill in the art.

[0102] In certain embodiments, the recombinant nucleic acid molecule is a plasmid. In certain embodiments, the recombinant nucleic acid molecule (e.g., a plasmid) is useful for rAAV production. In certain embodiments, the recombinant nucleic acid molecule (e.g., a plasmid) useful for rAAV production comprises a vector genome comprising 5'-ITR.CB.CI.IL2.V1.trastuzumab-coGW.SV40.3'-ITR. In certain embodiments, the recombinant nucleic acid molecule (e.g., a plasmid) useful for rAAV production comprises a vector genome comprising the nucleic acid sequence of SEQ ID NO:1. In certain embodiments, the recombinant nucleic acid molecule (e.g., a plasmid) useful for rAAV production comprises a vector genome comprising 5'-ITR.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40.3'-ITR. In certain embodiments, the recombinant nucleic acid molecule (e.g., a plasmid) useful for rAAV production comprises a vector genome comprising the nucleic acid sequence of SEQ ID NO:25. In certain embodiments, a recombinant nucleic acid molecule (e.g., a plasmid) useful for rAAV production comprises a vector genome comprising 5'-ITR.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40.3'-ITR. In certain embodiments, a recombinant nucleic acid molecule (e.g., a plasmid) useful for rAAV production comprises a vector genome comprising the nucleic acid sequence of SEQ ID NO:27.

[0103] Methods for generating and isolating AAV suitable for use as a vector are known in the art. See generally, for example, Grieger & Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10:717-733, and the references cited below, each of which is incorporated herein by reference in its entirety. As used herein, gene therapy vector refers to the rAAV described herein, suitable for use in treating patients. The ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the gene in order to package the gene into virions. The cap and rep genes can be supplied in trans.

[0104] In one embodiment, the expression cassettes described herein are engineered into genetic elements (e.g., shuttle plasmids) that introduce the immunoglobulin construct sequences carried therein into packaging host cells to produce viral vectors. In one embodiment, the selected genetic elements can be delivered to AAV packaging cells by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be generated. Methods used to generate such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0105] The term "AAV intermediate" or "AAV vector intermediate" refers to assembled rAAV capsids that lack the desired genomic sequence packaged therein. These may be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of the expression cassette or may contain only partially packaged genomic sequence that is insufficient to achieve expression of the gene product. These empty capsids are non-functional for introducing a gene of interest into a host cell.

[0106] The recombinant adeno-associated virus (AAV) described herein can be produced using known techniques. See, for example, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, US7588772B2. Such methods include culturing a host cell that contains an expression cassette consisting of a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, at a minimum an AAV inverted terminal repeat (ITR) and a transgene, and sufficient helper functions to allow packaging of the expression cassette into an AAV capsid protein. Methods for producing capsids, coding sequences therefor, and methods for producing rAAV viral vectors have been described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. USA100(10), 6081-6086(2003) and US2013 / 0045186A1.

[0107] In one embodiment, a producer cell culture is provided that is useful for producing a recombinant AAV having a capsid selected from AAVhu68, AAVrh91, AAVhu95, or AAVhu96. Such cell cultures comprise a nucleic acid that expresses an AAVhu68 capsid protein in a host cell (e.g., SEQ ID NO:37 or SEQ ID NO:38); a nucleic acid molecule suitable for packaging into an AAVhu68 capsid, e.g., a vector genome comprising a non-AAV nucleic acid sequence encoding a gene operably linked to AAV ITRs and a regulatory sequence that directs expression of the gene in a host cell; and sufficient AAV rep and adenovirus helper functions to allow packaging of the vector genome into a recombinant AAVhu68 or AAVrh91 capsid (e.g., SEQ ID NO:40 or SEQ ID NO:41), AAVhu95 capsid (e.g., SEQ ID NO:43 or SEQ ID NO:44), AAVhu96 capsid (e.g., SEQ ID NO:47 or SEQ ID NO:48). In one embodiment, the cell culture comprises a mammalian cell (e.g., human embryonic kidney 293 cells, among others) or an insect cell (e.g., Spodoptera frugiperda (Sf9) cells). In certain embodiments, the baculovirus provides the helper functions necessary for packaging the vector genome into recombinant AAVhu68, AAVrh91, AAVhu95, or AAVhu96 capsids.

[0108] Optionally, the rep function is provided by an AAV other than AAV2, selected to complement the source of the ITRs.

[0109] In one embodiment, the cells are produced in a suitable cell culture (e.g., HEK293 or Sf9) or suspension. The methods for producing gene therapy vectors described herein include methods well known in the art, such as production of plasmid DNA used for the production of gene therapy vectors, production of vectors, and purification of vectors. In some embodiments, the gene therapy vector is an AAV vector, and the produced plasmids are AAV cis-plasmids encoding the AAV vector genome and gene of interest, AAV trans-plasmids containing the AAV rep and cap genes, and adenovirus helper plasmids. The vector production process may include method steps such as initiation of cell culture, passaging of cells, seeding of cells, transfection of cells with plasmid DNA, medium exchange with serum-free medium after transfection, and harvesting of vector-containing cells and culture medium. The harvested vector-containing cells and culture medium are referred to herein as crude cell harvest. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems generally, see, e.g., Zhang et al., 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties. Methods of making and using these and other AAV production systems are also described in the following United States patents, the contents of each of which are incorporated herein by reference in their entirety: U.S. Pat. Nos. 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065.

[0110] The crude cell harvest may then be subjected to process steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector. Affinity chromatography purification followed by anion exchange resin chromatography is used to purify the vector drug product and remove empty capsids. These methods are described in more detail in WO2017 / 160360, filed December 9, 2016, entitled "Scalable Purification Method for AAV9" (incorporated by reference). A method for purifying AAV8: WO2017 / 100676, filed December 9, 2016, and a method for purifying rh10: WO2017 / 100704, filed December 9, 2016, entitled "Scalable Purification Method for AAVrh10" (also filed December 11, 2015), and a method for purifying AAV1: WO2017 / 100674, filed December 9, 2016, and "Scalable Purification Method for AAV1", filed December 11, 2015, are all incorporated herein by reference. Other suitable methods may be selected.

[0111] To calculate the content of empty and full particles, the VP3 band volume for a selected sample (e.g., in the examples herein, a preparation purified by iodixanol gradient, number of genome copies (GC) = number of particles) is plotted against the GC particles loaded. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the peak of the test sample. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL gives empty pt / mL. Empty pt / mL is divided by pt / mL and multiplied by 100 to obtain the percentage of empty particles.

[0112] In general, methods for assaying AAV vector particles containing empty capsids and packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves subjecting the processed AAV stock to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% Tris-acetate in buffer, then running the gel until the sample material is separated, blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. The anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used that binds to the primary antibody and includes a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody that contains a detection molecule covalently bound to the antibody, most preferably a sheep anti-mouse IgG antibody covalently bound to horseradish peroxidase. To semi-quantitatively determine the binding between the primary and secondary antibodies, a method for detecting binding is used, preferably a detection method capable of detecting radioisotope radiation, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins resolved in precast gradient polyacrylamide gels (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or other suitable staining methods, i.e., SYPRO Ruby or Coomassie staining.In one embodiment, the concentration of AAV vector genome (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). The samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using a TaqMan™ fluorogenic probe specific for the primers and the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. An endpoint assay based on digital PCR can also be used.

[0113] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serum protease, such as proteinase K (e.g., commercially available from Qiagen), is used. More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Suitably, the sample is diluted with an amount of proteinase K buffer equal to the sample size. The proteinase K buffer can be concentrated 2-fold or more. Typically, the proteinase K treatment is about 0.2 mg / mL, but can vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally carried out at about 55° C. for about 15 minutes, but may be performed at lower temperatures (e.g., about 37° C. to about 50° C.) for longer periods (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60° C.) for shorter periods (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95° C. for about 15 minutes, but may be performed at lower temperatures (e.g., about 70 to about 90° C.) and for longer periods (e.g., about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0114] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, for example, M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr; 25(2): 115-25. doi: 10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0115] In certain embodiments, the manufacturing processes for rAAV described herein involve methods described in U.S. Provisional Patent Application No. 63 / 371,597, filed August 16, 2022, and U.S. Provisional Patent Application No. 63 / 371,592, filed August 16, 2022, which are incorporated by reference in their entireties herein.

[0116] Briefly, a method for separating rAAVhu68 (or AAVrh91, AAVhu95, or AAVhu96) particles having packaged genomic sequences from genome-deficient AAVhu68 (or AAVrh91, or AAVhu95, or AAVhu96) intermediates comprises subjecting a suspension containing recombinant AAVhu68 (or AAVrh91) viral particles and AAVhu68 (or AAVrh91, or AVhu95, or AAVhu96) capsid intermediates to high performance liquid chromatography, where the AAVhu68 (or AAVrh91, or AAVhu95, or AAVhu96) viral particles and AAVhu68 intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 10.2 (or about 9.8 in the case of AAVrh91) and subjected to a salt gradient while monitoring the eluate for ultraviolet absorbance at about 260 nanometers (nm) and about 280 nm. The pH can range from about 10 to 10.4, although less optimal for rAAVhu68 and AAVrh91. In this method, AAV full capsids are recovered from fractions that are eluted when the A260 / A280 ratio reaches an inflection point. In one example, for an affinity chromatography step, the diafiltered product may be applied to an affinity resin (Life Technologies) that efficiently captures AAV serotypes. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, and AAV particles are efficiently captured.

[0117] The rAAV.trastuzumab-coGW (e.g., rAAV.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40, rAAV.UbC.PI.IL2.V1.trastuzumab-coGW.SV40, rAAV.CB7.CI.IL2_Furin_V1.trastuzumab-coGW.SV40, or rAAV.CB7.CI.IL2.V1.trastuzumab-coGW.SV40) is suspended in a suitable physiologically compatible composition (e.g., buffered saline). This composition can be frozen for storage and later thawed, and optionally diluted with a suitable diluent. Alternatively, the vector can be prepared as a composition suitable for delivery to a patient without undergoing a freezing and thawing step.

[0118] As used herein, the term "clade" in relation to a group of AAVs refers to a group of AAVs that are phylogenetically related to each other, as determined using a neighbor-joining algorithm based on an alignment of AAV vp1 amino acid sequences, with a bootstrap value of at least 75% (out of at least 1000 replicates) and a Poisson-corrected distance measure of 0.05 or less. Neighbor-joining algorithms have been described in the literature. See, for example, Nei and S. Kumar, Molecular Evolution and Phylogenetics, Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, as well as the sequence of the AAV vp1 capsid protein, one of skill in the art can readily determine whether a selected AAV is in one clade identified herein, in another clade, or outside these clades. See, for example, G Gao, et al, J Virol, 2004. Jun;78(10):6381-6388, which identifies clades A, B, C, D, E and F, and provides the nucleic acid sequences of novel AAVs, GenBank Accession Nos. AY530553-AY530629. See also WO2005 / 033321.

[0119] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to constructs in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, DM McCarty et al, "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference in its entirety.

[0120] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged into a viral capsid or envelope, and any viral genomic sequences packaged into the viral capsid or envelope are replication-defective, i.e., they cannot produce progeny virions, but can retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only the gene of interest flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0121] As used herein, the terms "recombinant AAV", "rAAV" and "artificial AAV" are used interchangeably and refer to, but are not limited to, AAV including a capsid protein and a vector genome packaged therein, the vector genome including a nucleic acid heterologous to AAV. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such an artificial capsid can be produced by any suitable technique using a selected AAV sequence (e.g., a fragment of vp1 capsid protein) in combination with a heterologous sequence that can be obtained from a different selected AAV that is a non-contiguous portion of the same AAV, from a non-AAV viral source, or from a non-viral source. An artificial AAV can be, but is not limited to, a pseudotyped AAV capsid, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. A pseudotyped vector in which the capsid of one AAV is replaced with a heterologous capsid protein is useful in the present invention. In one embodiment, AAV2 / 5 and AAV2 / 8 are exemplary pseudotype vectors. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. The methods used to create such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0122] Often, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endonucleases and exonucleases can be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded and / or double-stranded DNA, and RNA. Such steps can include a single nuclease, or a mixture of nucleases directed to different targets, which can be endonucleases or exonucleases.

[0123] The term "nuclease resistant" indicates that the AAV capsid is constructed entirely around an expression cassette designed to deliver genes into a host cell, and protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present from the production process.

[0124] As used herein, the term "heterologous" or any grammatical variation thereof when used in reference to vp capsid proteins refers to a population of non-identical members, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences.

[0125] The term "heterologous" as used in reference to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of vp1, vp2, and vp3 proteins within the capsid. AAV capsids contain subpopulations within vp1, vp2, and vp3 proteins that have predicted amino acid residue modifications. These subpopulations contain, at a minimum, specific deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, where deamidation results in amino acid changes and other optional modifications.

[0126] As used herein, a "subpopulation" of vp proteins refers to a group of vp proteins that have at least one defined common feature and that consists of at least one group member and fewer than all members of the reference group, unless otherwise specified. For example, a "subpopulation" of vp1 proteins, unless otherwise specified, is at least one vp1 protein and fewer than all vp1 proteins in an assembled AAV capsid. A "subpopulation" of vp3 proteins, unless otherwise specified, can be one vp3 protein and fewer than all vp3 proteins in an assembled AAV capsid. For example, in an assembled AAV capsid, vp1 protein can be a subpopulation of vp proteins, vp2 protein can be another subpopulation of vp proteins, and vp3 is yet another subpopulation of vp proteins. In another example, vp1, vp2, and vp3 proteins can include subpopulations having, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., different modifications at asparagine-glycine pairs.

[0127] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to the published literature, which provides general guidance to one of ordinary skill in the art for many of the terms used herein.

[0128] Pharmaceutical Compositions In one aspect, provided herein is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer.

[0129] In one embodiment, the rAAV is about 1×10 9 Genome copies (GC) / mL ~ approx. 1 x 10 14 In a further embodiment, the rAAV is formulated at about 3×10 9 GC / mL ~ approx. 3×10 13 In yet a further embodiment, the rAAV is formulated at about 1×10 9GC / mL ~ approx. 1×10 13 In one embodiment, the rAAV is formulated at a concentration of at least about 1×10 11 Formulated in GC / mL.

[0130] Also provided herein is a composition comprising the rAAV described herein and an aqueous suspension medium.In certain embodiments, the suspension is formulated for intravenous delivery, intrathecal administration, or intracerebroventricular administration.In one aspect, the composition contains at least one rAAV stock and optional carriers, excipients, and / or preservatives.

[0131] As used herein, a "stock" of rAAV refers to a population of rAAV. Despite the heterogeneity of capsid proteins due to deamidation, rAAV within a stock are expected to share the same vector genome. A stock may, for example, contain rAAV with capsids having selected AAV capsid proteins and heterogeneous deamidation patterns characteristic of a selected production system. A stock may be produced from a single production system or pooled from multiple runs of a production system. A variety of production systems may be selected, including but not limited to those described herein.

[0132] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmacologically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, nanoparticles, lipid nanoparticles (LNPs), microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivery vector genome can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, and the like.

[0133] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.Optionally, one or more surfactants are present in the formulation.In another embodiment, the composition can be delivered as a concentrate that is diluted for administration to a subject.In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0134] A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are non-toxic.

[0135] In one embodiment, the composition comprises a carrier, diluent, excipient and / or adjuvant. A suitable carrier can be easily selected by a person skilled in the art in view of the indication for which the introduced virus is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should contain a component that prevents rAAV from adhering to the injection tube but does not interfere with rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, can be selected from among non-toxic non-ionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated in a primary hydroxyl group is selected, such as Poloxamer 188 (also known under the trade names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, Kolliphor® P188) having a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e. non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycapric acid glyceride), polyoxy-oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 giving the approximate molecular mass of the polyoxypropylene core and the last digit x 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0136] In certain embodiments, compositions containing rAAV.trastuzumab-coGW are delivered at a pH ranging from 6 to 8, or 7.2 to 7.8, or 7.5 to 8. For intrathecal delivery, a pH greater than 7.5, e.g., 7.5 to 8, or 7.8, may be desirable. For intravenous delivery, a pH of about 6.8 to about 7.2 may be desirable.

[0137] In certain embodiments, the formulation may contain a buffered saline solution that does not contain sodium bicarbonate. Such a formulation may contain a buffered saline solution, such as Harvard buffer, which contains one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof in water. In one embodiment, the buffer is PBS. In another embodiment, the buffer is an artificial cerebrospinal fluid (aCSF), such as Elliot's formulation buffer, or Harvard apparatus perfusate (artificial CSF with final ionic concentrations (in mM): Na 150, K 3.0, Ca 1.4, Mg 0.8, P 1.0, Cl 155). The aqueous solution may further contain Kolliphor® P188, a poloxamer, which is commercially available from BASF and was previously sold under the trade name Lutrol® F68. The aqueous solution may have a pH of 7.2.

[0138] In another embodiment, the formulation may contain a buffered saline solution comprising 1 mM sodium phosphate (Na3PO4), 150 mM sodium chloride (NaCl), 3 mM potassium chloride (KCl), 1.4 mM calcium chloride (CaCl2), 0.8 mM magnesium chloride (MgCl2), and 0.001% poloxamer (e.g., Kolliphor®) 188, pH 7.2. See, e.g., harvardapparatus.com / harvard-apparatus-perfusion-fluid.html. In certain embodiments, Harvard buffer is preferred due to better pH stability observed with Harvard buffer.

[0139] In certain embodiments, the formulation buffer is an artificial CSF containing Pluronic F68. In other embodiments, the formulation may contain one or more penetration enhancers. Examples of suitable penetration enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0140] Optionally, the compositions of the invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0141] The composition according to the invention may include a pharma- ceutically acceptable carrier as defined above. Suitably, the composition described herein comprises one or more AAV suspended in an effective amount of a pharma- ceutically suitable carrier and / or mixed with suitable excipients designed for delivery to a subject via infusion, osmotic pump, intrathecal catheter, or for delivery by another device or route. In certain embodiments, an Ommaya reservoir is used for delivery. In one example, the composition is formulated for intrathecal delivery. In one example, the composition is formulated for intravenous (iv) delivery.

[0142] In one embodiment, the vector is included in a therapeutically effective amount in a pharmaceutical composition. The choice of carrier is not limiting of the present invention. Other conventional pharma- ceutically acceptable carriers, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0143] As used herein, the term "dose" or "amount" can refer to the total dose or amount delivered to a subject over the course of treatment, or the dose or amount delivered in a single unit (or multiple unit or divided dose) administration.

[0144] In certain embodiments, the composition comprises about 1×10 9 GC ~ Approximately 1 × 10 per gram (g) of brain mass 13 The rAAV can be formulated into dosage units containing amounts of rAAV in genome copy (GC) amounts (including all integers or fractions within the ranges and endpoints). In another embodiment, the dosage is 1×10 per gram of brain mass. 10 GC ~ approx. 1 × 10 per gram of brain mass 13 In certain embodiments, the dose of the vector administered to the patient is at least about 1.0×10 9 GC / g, approx. 1.5×10 9 GC / g, approx. 2.0×10 9 GC / g, approx. 2.5×10 9 GC / g, approx. 3.0×10 9 GC / g, approx. 3.5×10 9 GC / g, approx. 4.0×10 9 GC / g, approx. 4.5×10 9 GC / g, approx. 5.0×10 9 GC / g, approx. 5.5×10 9 GC / g, approx. 6.0×10 9 GC / g, approx. 6.5×10 9 GC / g, approx. 7.0×10 9 GC / g, approx. 7.5×10 9 GC / g, approx. 8.0×10 9 GC / g, approx. 8.5×10 9 GC / g, approx. 9.0×10 9 GC / g, approx. 9.5×10 9 GC / g, approx. 1.0×10 10 GC / g, approx. 1.5×10 10 GC / g, approx. 2.0×10 10 GC / g, approx. 2.5×10 10 GC / g, approx. 3.0×10 10GC / g, about 3.5×10 10 GC / g, about 4.0×10 10 GC / g, about 4.5×10 10 GC / g, about 5.0×10 10 GC / g, about 5.5×10 10 GC / g, about 6.0×10 10 GC / g, about 6.5×10 10 GC / g, about 7.0×10 10 GC / g, about 7.5×10 10 GC / g, about 8.0×10 10 GC / g, about 8.5×10 10 GC / g, about 9.0×10 10 GC / g, about 9.5×10 10 GC / g, about 1.0×10 11 GC / g, about 1.5×10 11 GC / g, about 2.0×10 11 GC / g, about 2.5×10 11 GC / g, about 3.0×10 11 GC / g, about 3.5×10 11 GC / g, about 4.0×10 11 GC / g, about 4.5×10 11 GC / g, about 5.0×10 11 GC / g, about 5.5×10 11 GC / g, about 6.0×10 11 GC / g, about 6.5×10 11 GC / g, about 7.0×10 11 GC / g, about 7.5×10 11 GC / g, about 8.0×10 11 GC / g, about 8.5×10 11 GC / g, about 9.0×10 11 GC / g, about 9.5×10 11 GC / g, about 1.0×10 12 GC / g, about 1.5×10 12 GC / g, about 2.0×10 12 GC / g, about 2.5×10 12 GC / g, about 3.0×10 12 GC / g, about 3.5×10 12 GC / g, about 4.0×10 12 GC / g, about 4.5×10 12 GC / g, about 5.0×10 12GC / g, approx. 5.5×10 12 GC / g, approx. 6.0×10 12 GC / g, approx. 6.5×10 12 GC / g, approx. 7.0×10 12 GC / g, approx. 7.5×10 12 GC / g, approx. 8.0×10 12 GC / g, approx. 8.5×10 12 GC / g, approx. 9.0×10 12 GC / g, approx. 9.5×10 12 GC / g, approx. 1.0×10 13 GC / g, approx. 1.5×10 13 GC / g, approx. 2.0×10 13 GC / g, approx. 2.5×10 13 GC / g, approx. 3.0×10 13 GC / g, approx. 3.5×10 13 GC / g, approx. 4.0×10 13 GC / g, approx. 4.5×10 13 GC / g, approx. 5.0×10 13 GC / g, approx. 5.5×10 13 GC / g, approx. 6.0×10 13 GC / g, approx. 6.5×10 13 GC / g, approx. 7.0×10 13 GC / g, approx. 7.5×10 13 GC / g, approx. 8.0×10 13 GC / g, approx. 8.5×10 13 GC / g, approx. 9.0×10 13 GC / g, approx. 9.5×10 13 GC / g, or approximately 1.0 x 10 14 GC / g brain mass.

[0145] The replication-deficient virus composition may be administered in a dose of approximately 1.0×10 for a human patient (to treat an average subject weighing 70 kg). 9 GC~approx. 1.0×10 16 A range of GC (including all integers or fractions within that range), preferably 1.0×10 12 GC~1.0×10 14 The dosage unit can be formulated to contain an amount of replication-deficient virus that is within the GC range. In one embodiment, the composition contains at least 1 x 10 per dose, including all integers or decimals within the range.9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9 x 10 9 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9 x 10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9 x 10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9 x 10 12 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×1013 , or 9 x 10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 14 , 2×10 14 , 3×10 14 , 4×1014, 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9 x 10 14 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9 x 10 15 In one embodiment, for human applications, the dose is formulated to contain 1×10 per dose, including all integers or fractions within the range. 10 ~Approx. 1×10 12 It may be in the GC range.

[0146] In certain embodiments, the composition is delivered intrathecally, optionally via intracisternal (ICM) injection. In certain embodiments, the composition is delivered via intraparenchymal administration. In certain embodiments, the composition is delivered via an Ommaya reservoir delivery system. In certain embodiments, the composition is delivered via direct injection into the tumor or tumor bed.

[0147] In one embodiment, a pharmaceutical composition comprising an rAAV described herein comprises about 1×10 9 GC ~ approx. 1 × 10 per gram of brain mass 13 It is administered in the dose of GC.

[0148] The rAAV, preferably suspended in a physiologically compatible carrier, can be administered to a human or non-human mammalian patient.

[0149] It is to be understood that the compositions in the pharmaceutical compositions described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0150] Use and Regimen In one aspect, the method provided herein is a method for treating a human subject diagnosed with HER2-positive cancer. The method comprises administering to the subject a suspension of rAAV vectors as described herein. In one embodiment, the method comprises administering to a subject with metastatic HER2-positive cancer in the brain a suspension of rAAV as described herein in a formulation buffer at about 1×10 per gram of brain mass. 9 GC ~ approx. 1 × 10 per gram of brain mass 14 In another embodiment, the method comprises administering to a subject a suspension of an rAAV described herein in a formulation buffer at a dose of about 1 x 10 per kg of body weight. 9 GC ~ approx. 1 x 10 per kg of body weight 14 Suitable doses for systemic and / or intratumoral administration can be determined by one skilled in the art.

[0151] In some embodiments, the method includes treating a subject diagnosed with a HER2-positive cancer that is a refractory and / or resistant cancer. In some embodiments, the method includes treating a subject diagnosed with a HER2-positive breast cancer that is a refractory and / or resistant cancer. In some embodiments, the method includes treating a subject diagnosed with a metastatic HER2-positive breast cancer in the brain that is a refractory and / or resistant cancer. In some embodiments, the method includes treating a subject diagnosed with a HER2-positive cancer that is trastuzumab resistant. In some embodiments, the method includes treating a subject with a metastatic HER2-positive breast cancer in the brain. In some embodiments, the method includes treating a subject diagnosed with a HER2-positive breast cancer that is trastuzumab resistant (e.g., estrogen receptor (ER) positive, progesterone receptor (PR) positive, and Her2 positive). In certain embodiments, the method includes treating a subject diagnosed with an ER-negative, PR-negative, and Her2-positive cancer. In certain embodiments, the method includes treating a subject diagnosed with a HER2 / low tumor.

[0152] As used herein, "refractory cancer" and / or "resistant cancer" refers to a cancer that is refractory or resistant to one or more cancer therapies, such as cancer chemotherapy (cytotoxic chemotherapy). In certain embodiments, a refractory and / or resistant cancer is not correctable with surgical intervention. In certain embodiments, a refractory and / or resistant cancer is initially unresponsive to chemotherapy or radiation therapy. In certain embodiments, a refractory and / or resistant cancer becomes unresponsive to cancer therapy over time.

[0153] As used herein, "trastuzumab resistance" refers to a cancer that is refractory or resistant to trastuzumab treatment. In certain embodiments, "refractory" or "resistant" means that the cancer (i.e., HER2 positive) is non-responsive to trastuzumab after a course of standard treatment, e.g., the cancer continues to progress even after trastuzumab treatment. In certain embodiments, the trastuzumab-resistant cancer is inherently resistant to trastuzumab treatment. In certain embodiments, the trastuzumab-resistant cancer acquires resistance, and the cancer cells initially respond to the treatment, but after some period of time, no longer respond to trastuzumab treatment (i.e., refractory to the treatment). In certain embodiments, the resistance of the HER2-positive tumor and non-responsive develops to late stage treatment or becomes resistant to trastuzumab therapy.

[0154] In certain embodiments, trastuzumab-resistant cancer cells are trastuzumab-sensitive, from parental cells that have been treated with a trastuzumab-containing composition and / or solution as a pretreatment or as a means of exerting selection pressure, as assessed in vitro. See also Pohlman, PR, et al., Resistance to Trastuzumab in Breast Cancer, 2009, Clin.Cancer Res.15(24):7479-7491; Vu, T., and Claret, FX, Trastuzumab: updated mechanisms of action and resistance in breast cancer, 2012, Front.Oncol.2(62); Rimawi, MF, et al., Resistance to Anti-HER2 Therapies in Breast Cancer, 2015, American Society of Clinical Oncology Educational Book 35(May 14, 2015)e157-e164, which are incorporated herein by reference.

[0155] In some embodiments, the method includes administering to a subject 1×10 suspension of a rAAV described herein in a formulation buffer. 11 ~1×10 14 In certain embodiments, the method comprises administering at a dose of about 1×10 GC / kg. 12 Genome copies (GC) / kg of rAAV ~ approximately 1 x 10 14 In certain embodiments, the dose is about 1×10 per patient. 13 GC / kg ~ approx. 1×10 14 GC rAAV, or approximately 3 × 10 13 In certain embodiments, intravenous delivery is about 3×10 12 GC / kg ~ approx. 1×10 14 GC / kg, approximately 3.0 × 10 13 GC / kg and approximately 1.0 x 10 13 Further included is the dose of GC / kg.

[0156] In one embodiment, the subject is delivered a therapeutically effective amount of the rAAV described herein. As used herein, "therapeutically effective amount" refers to the amount of a composition comprising a nucleic acid sequence encoding a Trastuzuma immunoglobulin that delivers and expresses a sufficient amount of immunoglobulin to target cells to achieve efficacy. The dosage is adjusted to balance the therapeutic effect against any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is used. The expression level of the transgene product can be monitored to determine the frequency of dosage that results in a viral vector, preferably an AAV vector containing a transgene. Optionally, a similar dosage regimen as described for therapeutic purposes can be utilized for immunization using the compositions provided herein.

[0157] Suitable conventional pharma- ceutically acceptable routes of administration include, but are not limited to, the desired organ (e.g., brain, CSF, heart), intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, intraparenchymal, intraventricular, intrathecal, ICM, lumbar puncture, and other parenteral routes of administration. Routes of administration may be combined, if desired.

[0158] Suitable volumes for delivery of these doses and concentrations can be determined by one of skill in the art. For example, a volume of about 1 μL to 150 mL can be selected for CNS delivery, with higher volumes selected for adults. Typically, for newborns, a suitable volume can be selected from about 0.5 mL to about 10 mL, and for older infants, from about 0.5 mL to about 15 mL. For toddlers, a volume of about 0.5 mL to about 20 mL can be selected. For children, a volume of up to about 30 mL can be selected. For preteens and teens, a volume of up to about 50 mL can be selected. In yet other embodiments, patients can receive intrathecal administration with a selected volume of about 5 mL to about 15 mL, or about 7.5 mL to about 10 mL. Other suitable volumes and dosages can be determined for CNS delivery, intratumoral delivery, and / or systemic delivery (e.g., IV). Dosages are adjusted to balance therapeutic benefit against any side effects, and such dosages can vary depending on the therapeutic application for which the recombinant vector is used.

[0159] In one embodiment, the method comprises administering the rAAV described herein to a subject in need thereof. The recombinant vectors described above may be administered or delivered to a host cell according to published methods. The rAAV, preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient subject. In certain embodiments, for administration to a human patient, the rAAV is preferably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. Preferably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6-9, or pH 6.5-7.5, pH 7.0-7.7, or pH 7.2-7.8. The pH of cerebrospinal fluid is about 7.28 to about 7.32, so for intrathecal delivery, a pH within this range may be desirable, and for intravenous delivery, a pH of about 6.8 to about 7.2 may be desirable. For intravenous delivery, a pH of about 6.8 to about 7.2 may be desirable. However, other pH's within the broadest range, and subranges thereof, may be selected for other delivery routes.

[0160] Suitably, the pharmaceutical compositions and uses thereof described herein include delivery to a subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route. In one example, the composition is formulated for intrathecal delivery.

[0161] As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to a route of administration of a drug via injection into the spinal canal, more specifically, via injection into the subarachnoid space to reach the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intraventricular lateral cerebroventricular (ICV)), suboccipital / intracisternal, and / or C1-2 puncture. For example, the material may be introduced by means of a lumbar puncture for diffusion throughout the subarachnoid space. Another example may be an injection into the cisterna magna. In certain embodiments, the rAAV, vector, or composition described herein is administered to a subject in need thereof via intrathecal administration. In certain embodiments, intrathecal administration is performed as described in U.S. Patent Publication No. 2018 / 0339065A1, published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, CNS administration is performed using an Ommaya reservoir (also referred to as an Ommaya device or Ommaya system).

[0162] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to the route of administration of a drug directly into the cerebrospinal fluid of the cerebellum-medullary cisterna magna, more specifically, by suboccipital puncture or by direct injection into the cisterna magna, or by a permanently placed tube.

[0163] As used herein, the terms "intracembrane," "dentate nucleus," or IDN refer to a route of administering a composition directly to the dentate nucleus. IDNs allow for targeting of the dentate nucleus and / or cerebellum. In certain embodiments, IDN administration is performed using the ClearPoint® Neuro Navigation System (MRI Interventions, Inc., Memphis, TN) and a ventricular cannula, which allows for MRI-guided visualization and administration. Alternatively, other devices and methods may be selected.

[0164] Suitably, the pharmaceutical compositions described herein and their uses include delivery to a subject to a tumor bed via surgical and non-surgical techniques. See also Gilbert, A., and Machluf, M., Nano to micro delivery systems: targeting angiogenesis in brain tumors, J Angiogenes Res. 2010 Oct 8; 2(1): 20; Balyasnikova, IV., et al., Intranasal Delivery of Mesenchymal Stem Cells Significantly Extends Survival of Irradiated Mice with Experimental Brain Tumors, Molecular Therapy, 2014, 22(1): P140-P148, epub January 1, 2014, which are incorporated herein by reference in their entirety. In certain embodiments, the pharmaceutical compositions described herein and their uses include delivery directly to a tumor or tumor bed in the CNS. In certain embodiments, the pharmaceutical compositions described herein and their uses include delivery via a systemic route. In certain embodiments, the pharmaceutical compositions and uses thereof described herein include direct delivery to Her2-positive tumors located outside the CNS (e.g., breast cancer or metastatic Her2-positive cancer, or gastric gastroesophageal junction cancer). See also Ma, H., et al., Intratumoral gene therapy of malignant brain tumor in a rat model with angiostatin delivered by adeno-associated viral (AAV) vector, 2002, Gene Therapy, 9:2-11; Liang, C., et al.,Local Expression of Secondary Lymphoid Tissue Chemokine Delivered by Adeno-Associated Virus within the Tumor Bed Stimulates Strong Anti-Liver Tumor Immunity,Journal of Virology,2007,81(17):9502-9511,Xu,X.,et al.,Adeno-associated virus(AAV)-based gene therapy for glioblastoma,Cancer Cell International,2021,21:76,epub 26 January 2021,Reul,J.,et al.,Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors,Methods(Frontiers in Oncology),2019,9:52,epub February 14,2019,Sheth,RA.,et al.,Assessment of Image-Guided Intratumoral Delivery of Immunotherapeutics in Patients With See, Cancer, JAMA Netw Open. 2020;3(7):e207911, epub July 29, 2020; Tchou, J., et al., Safety and Efficacy of Intratumoral Injections of Chimeric Antigen Receptor (CAR) T Cells in Metastatic Breast Cancer, Cancer Immunol Res, 2017,5(12):1152-1161, epub November 6, 2017, all of which are incorporated by reference in their entirety.

[0165] In a further embodiment, the compositions of the invention are preferably designed such that the rAAV vector carries a nucleic acid expression cassette encoding a trastuzumab immunoglobulin construct and a regulatory sequence that directs the expression of the trastuzumab immunoglobulin in selected cells. After administration of the vector to the CNS, the vector delivers the expression cassette to the CNS and expresses the proteinaceous immunoglobulin construct in vivo. The use of the compositions described herein in anti-neoplastic methods, as well as the use of these compositions in anti-neoplastic regimens, can optionally be accompanied by the delivery of one or more other anti-neoplastic or other active agents.

[0166] As mentioned above, the composition may contain a single type of AAV vector described herein, which includes an expression cassette for delivering an anti-neoplastic trastuzumab immunoglobulin construct in vivo. Alternatively, the composition may contain two or more different AAV vectors, each of which has a different expression cassette packaged therein. For example, two or more different AAVs may have different expression cassettes expressing immunoglobulin polypeptides that assemble in vivo to form a single functional immunoglobulin construct. In another example, two or more AAVs may have different expression cassettes expressing immunoglobulin polypeptides for different targets, for example, two provide two functional immunoglobulin constructs (e.g., an anti-Her2 immunoglobulin construct and a second anti-neoplastic immunoglobulin construct).

[0167] The regimens described herein may include, in addition to one or more of the combinations described herein, further combinations with one or more of anti-tumor biological drugs, anti-tumor small molecule drugs, chemotherapeutic agents, immune enhancing agents, radiation, surgery, etc. The biological drugs described herein are based on peptides, polypeptides, proteins, enzymes, nucleic acid molecules, vectors (including viral vectors), etc.

[0168] In one embodiment, the method further comprises subjecting the subject to immunosuppressive combination therapy. Immunosuppressants for such combination therapy include, but are not limited to, glucocorticoids, corticosteroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalogs), and cytostatic agents (including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins). Immunosuppressants may include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25)-specific antibodies or CD3-specific antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-alpha) binding agents.

[0169] In certain embodiments, immunosuppressive therapy may be initiated 0, 1, 2, 7 days, or earlier prior to gene therapy administration. Such therapy may include simultaneous administration of two or more drugs on the same day, such as prednerisone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin). One or more of these drugs may be continued at the same dose or at adjusted doses after gene therapy administration. Such therapy may be for about 1 week (7 days), about 60 days, or more, as needed. In certain embodiments, a regimen that does not include tacrolimus is selected.

[0170] In one embodiment, the method further comprises administering an anti-AAV neutralizing antibody (NAb) to the subject to reduce peripheral transduction and mitigate the potential risk of trastuzumab-induced cardiotoxicity. In certain embodiments, the method further comprises detecting the presence of systemic AAV NAb prior to treatment with the anti-AAV NAb, and patients with a level of anti-AAV NAb above a predetermined level against the rAAV capsid (or serum cross-reactive capsid) do not require pretreatment. Such a level may be, for example, above about 1:10, about 1:20, about 1:50, about 1:100, about 1:250, or a higher or lower level. In certain embodiments, the method further includes intravenously administering to the patient a human anti-AAV polyclonal antibody (e.g., plasma-derived pooled human immunoglobulin (IVIG)), an anti-AAV monoclonal antibody, or a cocktail of anti-AAV antibodies about 1 day to about 2 hours prior to treatment with rAAV-trastuzumab, e.g., rAAV.trastuzumab-coGW.

[0171] In certain embodiments, a combination regimen is provided to prevent off-target delivery of rAAV, the regimen including (a) pretreating the patient by systemically administering a composition comprising an anti-AAV capsid neutralizing antibody directed against the AAV capsid in the recombinant AAV vector, and (b) administering a central nervous system (CNS) rAAV (e.g., rAAV.trastuzumab-coGW) described herein. See also U.S. Provisional Patent Application No. 63 / 328,227, filed April 6, 2022, which is incorporated by reference in its entirety.

[0172] As used herein, a "neutralizing antibody" or "NAb" specifically binds to a viral capsid or envelope and interferes with the infectivity of a virus or a recombinant viral vector having a viral capsid or envelope, thus preventing the recombinant viral vector from delivering an effective amount of a gene product encoded by an expression cassette in its vector genome. A variety of methods can be utilized to assess neutralizing antibodies in a patient's serum. The terms method and assay can be used interchangeably. As used herein, the terms "neutralization assay" and "serum virus neutralization assay" refer to a serological test to detect the presence of systemic antibodies that can interfere with the infectivity of a virus. Such assays can also qualitatively or quantitatively identify the binding ability (e.g., magnitude) or efficiency of an antibody to neutralize a target. Immunoassays may include enzyme immunoassays (EIAs), radioimmunoassays (RIAs) using radioisotopes, fluorescent immunoassays (FIAs) using fluorescent materials, chemiluminescent immunoassays (CLIAs) using chemiluminescent materials, as well as counting immunoassays (CIAs) employing particle counting techniques, Western blots, immunohistochemistry (IHC), and other modified assays such as agglutination. One of the most common enzyme immunoassays is the enzyme-linked immunosorbent assay (ELISA).

[0173] Examples of suitable methods include those described, for example, in R Calcedo, et al, Journal Infectious Diseases, 2009, 199: 381-290; GUO, et al., "Rapid AAV_Neutralizing Antibody Determination with a Cell-Binding Assay", Molecular Therapy: Methods & Clinical Development Vol. 13 June 2019; T. Ito et al, "A convenient enzyme-linked immunosorbent assay for rapid screening of anti-adeno-associated virus neutralizing antibodies", Ann Clin Biochem 2009; 46: 508-510; US2018 / 0356394A2 (Voyager Therapeutics). In addition, commercially available kits exist (see, for example, Athena Diagnostics, Invitrogen, ThermoFisher.com, Covance).

[0174] The neutralizing ability of an antibody is usually measured via the expression of a reporter gene, such as luciferase or GFP. To determine and compare the activity of neutralizing antibodies, the antibody being tested should exhibit 50% or more neutralizing activity in one of the neutralization assays described herein. In some examples, the neutralizing ability is determined by measuring the activity of a reporter gene product (e.g., luciferase, GFP). The neutralizing ability of an antibody against a particular viral vector can be at least 50%, e.g., at least 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

[0175] As used herein, the term "NAb titer" is a measure of how many neutralizing antibodies (e.g., anti-AAV Nabs) that neutralize the physiological effect of the targeted epitope (e.g., AAV) are produced. Anti-AAV NAb titers can be measured, for example, as described in Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009.199(3):p.381-390, which is incorporated herein by reference.

[0176] Still other combination therapeutic agents may include, for example, anti-IgG enzymes, which have been reported to be useful in depleting anti-AAV antibodies (thus allowing administration to patients who test above a threshold level of antibodies to a selected AAV capsid), and / or one or more of a) a steroid or combination of steroids, and / or (b) an IgG cleaving enzyme, (c) an inhibitor of Fc-IgE binding, (d) an inhibitor of Fc-IgM binding, (e) an inhibitor of Fc-IgA binding, and / or (f) gamma interferon. Anti-FcRN antibodies include, for example, rozanolixizumab (UCB7665) (UCB SA), IMVT-1401, RVT-1401 (HL161), HBM9161 (all forms HanAll BioPhrma Co.Ltd), nipocalimab (M281) (Momenta Pharmaceuticals Inc), ARGX-113 (efgartigimod) (Argenx SE), orilanolimab (ALXN1830, SYNT001, Alexion Pharmaceuticals Inc), SYNT002, ABY-039 (Affibody AB), or DX-2507 (Takeda Pharmaceutical Co.Ltd). In certain embodiments, a combination of anti-FcRN antibodies is administered. In certain embodiments, an anti-FcRN antibody is administered in combination with a suitable anti-FcRn ligand (i.e., a peptide or protein construct that binds to human FcRn to inhibit IgG binding).

[0177] In one embodiment, a combination regimen for treating a patient with a Her2 positive tumor or a Her2 positive metastatic tumor is provided, the regimen comprising administering a vector described herein in combination with a ligand that inhibits binding of human FcRn and pre-existing patient neutralizing antibodies (e.g., IgG). In certain embodiments, the patient may be naive to any therapeutic treatment with the vector and may have pre-existing immunity due to prior infection with wild-type virus. In other embodiments, the patient may have neutralizing antibodies as a result of prior treatment or vaccination. In certain embodiments, the patient may have neutralizing antibodies of 1:1 to 1:20, or greater than 1:2, greater than 1:5, greater than 1:10, greater than 1:20, greater than 1:50, greater than 1:100, greater than 1:200, greater than 1:300, or higher. In certain embodiments, the patient has neutralizing antibodies in the range of 1:1 to 1:200, or 1:5 to 1:100, or 1:2 to 1:20, or 1:5 to 1:50, or 1:5 to 1:20. In certain embodiments, patients receive a single anti-FcRn ligand (e.g., an anti-FcRn antibody) as the only agent that regulates FcRn-IgG binding and enables effective vector delivery. In other embodiments, patients may receive a combination of one or more anti-FcRn ligands and a second component (e.g., an Fc receptor down-modulator (e.g., interferon gamma), an IgG enzyme, or another suitable component). Such combinations may be particularly desirable for patients with particularly high neutralizing antibody levels (e.g., greater than 1:200).

[0178] In certain embodiments, anti-FcRn ligand(s) (e.g., antibody) are administered to patients with neutralizing antibodies prior to, and optionally simultaneously with, the selected viral vector. In certain embodiments, continued expression of anti-FcRn ligand after administration of gene therapy vector may be desired on a short-term (transient basis), for example, until such time as the viral vector is cleared from the patient. In certain embodiments, sustained expression of anti-FcRn ligand may be desired. Optionally, in this embodiment, the ligand may be delivered via a viral vector, including, for example, within a viral vector expressing a therapeutic transgene. However, this embodiment is not desirable when the therapeutic gene being delivered is an antibody or antibody construct, or another construct that includes an IgG chain. In such embodiments, where an antibody construct with an IgG chain is delivered via a viral vector to a patient with pre-existing immunity, the anti-FcRn ligand is delivered or dosed transiently such that the amount of circulating anti-FcRn ligand is cleared from the serum before effective levels of the vector-mediated transgene product are expressed.

[0179] In certain embodiments, the FcRn ligand is delivered 1-7 days prior to administration of the vector (e.g., rAAV). In certain embodiments, the FcRn ligand is delivered daily. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered on the same day that the vector is administered. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered at least 1 day to 4 weeks after rAAV administration. In certain embodiments, the ligand is delivered 4 weeks to 6 months after rAAV administration. In certain embodiments, the ligand is administered via a different route of administration than the rAAV. In certain embodiments, the ligand is administered orally, intravenously, or intraperitoneally. See also International Patent Application No. PCT / US2021 / 037575, filed June 16, 2021, and now published WO2021 / 257668A1, both of which are incorporated by reference in their entireties.

[0180] In one embodiment, the compositions described herein are used in a method for slowing tumor growth, wherein the tumor is a metastatic HER2+ cancer in the brain. In one embodiment, the compositions described herein are used in a method for slowing tumor growth, wherein the tumor is a metastatic HER2+ breast cancer in the brain. In one embodiment, the compositions described herein are used in a method for slowing tumor growth, wherein the tumor is a metastatic HER2+ gastrointestinal cancer in the brain. In yet another embodiment, the compositions described herein are useful for reducing tumor size in a subject. In a further embodiment, the compositions described herein are useful for reducing the number of cancer cells in non-solid tumor cancers. In another embodiment, the compositions described herein are useful for prophylaxis. In one embodiment, the compositions described herein are used in a method for preventing the development of metastases in a patient having a HER2+ tumor, wherein the patient is at risk of developing metastases. In one embodiment, the compositions described herein are used in a method for preventing the development of brain metastases in a patient having an advanced HER2+ tumor, wherein the patient is at risk of developing brain metastases. In one embodiment, the compositions described herein are used in a method for preventing the development of brain metastases in patients with dHER2+ tumors after stereotactic radiosurgery, i.e., preventing recurrence in patients with HER2+ breast cancer brain metastases. In another embodiment, the compositions provided herein are used in a method for increasing overall survival and / or progression-free survival in patients. In certain embodiments, the compositions described herein are used in a method for treatment to delay the progression of brain metastases in patients who have achieved stable disease with standard therapy. In certain embodiments, the compositions described herein are used in a method for treatment to manage and alleviate symptoms of brain metastases in patients who are progressing. Optionally, the AAV compositions described herein are administered in the absence of additional exogenous pharmacological or chemical agents or other physical disruption of the blood-brain barrier.

[0181] In combination therapy, the AAV-delivered immunoglobulin constructs described herein are administered before, during, or after initiating therapy with another agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during, and after initiating anti-neoplastic therapy. For example, AAV can be administered 1-30 days, preferably 3-20 days, more preferably 5-12 days before initiating radiation therapy. In another embodiment of the invention, chemotherapy is administered simultaneously with, or more preferably after, AAV-mediated immunoglobulin (antibody) therapy. In yet other embodiments, the compositions of the invention can be combined with other biologics, such as recombinant monoclonal antibody drugs, antibody-drug conjugates, and the like. Additionally, combinations of different AAV-delivered immunoglobulin constructs as discussed above can be used in such regimens.

[0182] Any suitable method or route can be used to administer the AAV-containing compositions described herein, and optionally co-administer anti-neoplastic agents and / or antagonists of other receptors. The anti-neoplastic agent regimens utilized in accordance with the present invention include any regimen believed to be optimally suitable for treating the patient's neoplastic condition. Different malignancies may require the use of specific anti-tumor antibodies and specific anti-neoplastic agents, which will be determined on a patient-to-patient basis. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The dose of antagonist administered will depend on many factors, including, for example, the type of antagonist, the type and severity of the tumor being treated, and the route of administration of the antagonist.

[0183] kit In certain embodiments, kits are provided that include concentrated vector suspended in a formulation (optionally frozen), an optional dilution buffer, and devices and components required for intrathecal, intraventricular, or intracisternal administration. In another embodiment, the kit may additionally or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow injection. Such buffer may allow for about 1:1 to 1:5 dilution of the concentrated vector, or more. In other embodiments, more or less buffer or sterile water is included to allow for dose titration and other adjustments by the treating physician. In yet other embodiments, the kit includes one or more components of the device. Suitable dilution buffers are available, such as saline, phosphate buffered saline (PBS), or glycerol / PBS.

[0184] It is to be understood that the compositions in the kits described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0185] Devices and methods for delivery of pharmaceutical compositions In one aspect, the vectors, rAAVs, or compositions thereof provided herein may be administered intrathecally via the methods and / or devices provided in this section and described in WO2017 / 136500 and WO2018 / 160582 (incorporated herein by reference). Alternatively, other devices and methods may be selected. In certain embodiments, the method includes a step of CT-guided suboccipital injection into the patient's cisterna magna via a spinal needle. As used herein, the term computed tomography (CT) refers to radiography in which a three-dimensional image of a body structure is constructed by a computer from a series of planar cross-sectional images made along an axis. In certain embodiments, the device is described in U.S. Patent Publication No. 2018-0339065A1, published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, the vectors, rAAVs, or compositions thereof provided herein may be administered using an Ommaya reservoir.

[0186] It is to be understood that the compositions in the devices described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0187] As used herein, the phrases "alleviate symptoms," "ameliorate symptoms," or grammatical variations thereof, refer to the reversal of metastatic Her2+ cancer in brain-related symptoms. In one embodiment, alleviation or amelioration refers to the total number of symptoms in a patient after administration of a described composition(s) or use of a described method being reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to before administration or use. In another embodiment, alleviation or amelioration refers to the severity or progression of symptoms after administration of a described composition(s) or use of a described method being reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to before administration or use.

[0188] As used herein, "conservative amino acid replacement" or "conservative amino acid substitution" refers to the modification, replacement, or substitution of an amino acid with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size) known by those skilled in the art. Also see, for example, FRENCH et al. What is a conservative substitution? Journal of Molecular Evolution, March 1983, Volume 19, Issue 2, pp 171-175, and YAMPOLSKY et al. The Exchangeability of Amino Acids in Proteins, Genetics. 2005 Aug; 170(4): 1459-1472 (each of which is incorporated herein by reference in its entirety).

[0189] As used herein, the term "administering" or any grammatical variations thereof refers to the delivery of a composition described herein to a subject.

[0190] The terms "comprise", "comprises", and "comprising" should be interpreted inclusively rather than exclusively. The terms "consist", "consisting", and variations thereof should be interpreted exclusive rather than inclusive. Although various embodiments herein are set forth using the word "comprising", in other circumstances, the relevant embodiment is also intended to be included and described using the words "consisting of" or "consisting essentially of". As used throughout this specification and claims, the terms "comprising", "containing", "including", and variations thereof include other components, elements, integers, steps, etc. Conversely, the term "consisting" and variations thereof exclude other components, elements, integers, steps, etc.

[0191] It should be noted that the terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0192] As used herein, the term "about" or "~" refers to a ±10% variation from the reference integer and values ​​therebetween, unless otherwise specified. For example, "about" 500 μM includes ±50 (i.e., 450 to 550, including integers therebetween). For other values, particularly when referring to percentages (e.g., 90% of the value), the term "about" encompasses all within the range, including both integers and fractions.

[0193] As noted above, the term "about" when used to adjust a numerical value means a variation of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or any value therebetween) from the given reference, unless otherwise specified.

[0194] In certain cases, the terms "E+#" or "e+#" are used to refer to the exponent. For example, "5E10" or "5e10" refers to 5 × 10 10 These terms may be used interchangeably.

[0195] With regard to the description of various embodiments herein, it is contemplated that each of the compositions described herein is, in another embodiment, useful in the methods of the invention. In addition, each of the compositions described herein that are useful in the methods is, in another embodiment, also contemplated to be an embodiment of the invention itself.

[0196] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published documents which provide such persons with general guidance to many of the terms used in this application. EXAMPLES

[0197] The following examples are provided to illustrate certain aspects of the claimed invention, but the invention is not limited to these examples.

[0198] Breast cancer is the most common cause of brain metastases in women, affecting 15-30% of all patients. Despite the success of systemic Her2-targeted therapies, up to half of patients with Her2-positive (HER2-positive, HER2+, or her2+) primary breast cancer will develop breast cancer brain metastasis (BCBM). Subtype conversion from Her2- primary breast tumors accounts for an additional 15% of Her2+ BCBM, with the total of Her2-positive in the BCBM setting reaching nearly 65% ​​of cases. HER2-positive BCBM may manifest in the leptomeninges (LM) or parenchyma. LM lesions have direct access to the cerebrospinal fluid (CSF). LM disease accounts for 8% of total cases of HER2-positive BCBM. Despite the fact that direct delivery of Her2-targeted therapies to the central nervous system (CNS) has shown promising clinical results, long-term feasibility is limited, at least in part, by the need for repeated dosing to overcome shorter drug half-lives in the cerebrospinal fluid versus systemic circulation (e.g., approximately 1 vs. 5 days, respectively).

[0199] Trastuzumab, also called Herceptin, is a humanized monoclonal antibody (mAb) against HER2, developed by Genentech and commercialized by Genentech / Roche. Trastuzumab is indicated for the treatment of HER2+ breast cancer and metastatic breast cancer, both in combination with chemotherapy or as a single agent (after receiving chemotherapy). Trastuzumab plus chemotherapy increases both progression-free survival and overall survival in patients with metastatic breast cancer. Systemic administration of trastuzumab is not indicated for the treatment of BCBM because it cannot cross the blood-brain barrier.

[0200] To achieve sustained CNS expression of trastuzumab (Herceptin) without frequent dosing, we developed a gene therapy approach to treat BCBM that involves a single local administration of an AAV vector encoding trastuzumab. Previous studies have shown that intracerebroventricular (ICV) injection of trastuzumab-expressing AAV doubled the average lifespan of Rag1 knockout (KO) mice bearing BT-474 breast cancer brain tumors. Rag1 KO mice that received AAV-trastuzumab prior to brain implantation of BT-474 tumor cells showed a dose-dependent delay in tumor formation. See also International Patent Application No. PCT / US2015 / 027491, filed April 24, 2015, published October 29, 2015 as WO2015 / 164723A1.

[0201] In this study, we performed extensive gene therapy vector engineering and in vivo evaluation. Trastuzumab concentrations achieved in the brain parenchyma following a single ICV administration of the engineered vector in Rag1 KO mice ranged from 10 to 165 ng / mg brain protein by day 28.

[0202] Previous trials utilizing CNS delivery of gene therapy vectors demonstrated vector leakage into the systemic circulation, resulting in detectable transgene expression in the blood following peripheral transduction. To mitigate the potential risk of trastuzumab-induced cardiotoxicity, we investigated the use of intravenously administered, plasma-derived, pooled human immunoglobulin (IVIG) as a source of anti-AAV neutralizing antibodies to reduce peripheral transduction. Compared to controls receiving naive mouse serum, Rag1 KO mice that received IVIG 2 h prior to ICV administration of AAV-trastuzumab exhibited similar levels of trastuzumab in the brain while showing a 10-fold reduction in serum trastuzumab concentrations. Vector biodistribution analysis suggested that the reduced serum trastuzumab levels were due to an effective blockade of liver transduction following IVIG pretreatment.

[0203] These data suggest that administration of AAV vectors to the CNS that achieve sustained expression of trastuzumab may effectively treat Her2+ BCBM. In addition, patients with pre-existing neutralizing antibodies to the AAV vector capsid need not be excluded from future clinical trials.

[0204] Example 1: Production of rAAV containing engineered trastuzumab In the studies herein, various engineered sequences encoding trastuzumab were generated and comparative studies were performed. rAAV was generated using a triple transfection technique, utilizing (1) a cis plasmid encoding the AAV2 rep protein and the AAVhu68 VP1 cap gene (alternatively, AAVrh91, AAVhu95, or AAV9), (2) a cis plasmid containing adenovirus helper genes not provided by the packaging cell line expressing adenovirus E1a, and (3) a trans plasmid containing a vector genome for packaging into an AAV capsid. See, for example, US2020 / 0056159. The trans plasmid is designed to contain either a vector genome containing an engineered trastuzumab sequence including an IL-2 signal peptide and a T2A element, optionally including a furin cleavage sequence. The vector genome includes (among others) (i) (SEQ ID NO:1), (ii) (SEQ ID NO:25), (iii) (SEQ ID NO:27), or (iv) (SEQ ID NO:50).

[0205] The vector genome contains an AAV 5' inverted terminal repeat (ITR) and an AAV 3' ITR at the extreme 5' and 3' ends, respectively. The ITRs flank the sequence of an expression cassette packaged into the AAV capsid that contains the sequence encoding trastuzumab. The expression cassette further comprises regulatory sequences operably linked to the fusion protein coding sequence, including a promoter (either a chicken beta actin promoter or a CB7 hybrid promoter containing a chimeric intron containing a CMV IE enhancer, a chicken beta actin promoter, and a chicken beta actin intron, or a ubiquitin C (UbC) promoter), an intron (either a chicken beta actin intron or a chimeric intron containing a Promega intron), and an SV40 polyA.

[0206] AAV vector technology allows for sufficient expression of engineered trastuzumab transgenes in the brain when directed using various promoters. Expression of trastuzumab from AAV vectors provides an effective one-time gene therapy approach for the prevention and / or treatment of HER2+ BCBM. The present treatment strategy for HER2+ BCBM uses different combinations of HER2 modulators (anti-HER2 monoclonal antibodies and kinase inhibitors) and chemotherapeutic agents. Furthermore, intracranial delivery of AAV-trastuzumab in HER2+ breast cancer patients at high risk for the development of BCBM can prevent or significantly delay the appearance of tumor lesions. CNS delivery of AAV-trastuzumab (engineered trastuzumab coding sequence) can be used as an alternative approach to achieve disease remission or stabilization, either alone or in combination with standard of care systemic HER2 kinase inhibitors, as it can circumvent the treatmentscape and result in long-lasting trastuzumab expression.

[0207] Example 2: rAAV Evaluation in Mice Introduction The human epidermal growth factor receptor (EGFR) tyrosine kinase family is composed of four closely related membrane-bound cell surface receptors: EGFR, HER2, HER3, and HER4. Ligand-dependent homo- or heterodimerization of these receptors leads to phosphorylation of the intracellular tyrosine kinase domain and activation of signaling pathways that promote cell proliferation, migration, and differentiation. Among the EGFR family proteins, HER2 is the only constitutively active orphan receptor with no known ligand, and HER2 also has the strongest catalytic activity and is the preferred dimerization partner for all other members of the EGFR family. HER2 expression is required for normal mammalian embryonic development, but aberrant HER2 signaling is a known oncogenic driver. HER2 amplification / overexpression promotes tumorigenesis by overactivating the oncogenic RAS-MAPK and PI3 / AKT / mTOR signaling pathways. HER2 has been found to be amplified / overexpressed in several types of tumors (e.g., breast, gastric, esophageal, and ovarian cancer) and is often associated with an aggressive tumor phenotype and a higher risk of recurrence.

[0208] HER2 positivity is observed in 15-20% of all invasive breast cancer cases. Up to half of patients with HER2+ breast cancer go on to develop breast cancer brain metastases (BCBM). Subtype conversion arising from HER2- primary breast tumors accounts for an additional 15% of HER2+ BCBM, bringing the total HER2 positivity in the BCBM setting to nearly 65% ​​of cases. Targeting advanced HER2+ breast cancer using regimens including the anti-HER2 humanized monoclonal antibody trastuzumab (Herceptin®) has been associated with a marked increase in survival (currently approximately 5 years versus 1.5 years in 2001), but the median overall survival of patients diagnosed with HER2+ BCBM is only 29.1 months despite the most recent treatment (trastuzumab + tucatinib (HER2 tyrosine kinase inhibitor) + capecitabine). This discrepancy may be explained, at least in part, by the presence of an intact blood-brain barrier (420:1 ratio of trastuzumab levels in serum to cerebrospinal fluid (CSF)) that blocks large molecular weight biologics such as trastuzumab from entering the brain parenchyma. Due to subtherapeutic drug concentrations in the brain, HER2+ breast cancer patients receiving systemic antibody therapy are at increased risk of developing BCBM.

[0209] Alternatively, delivery of HER2-targeted therapy directly to the central nervous system (CNS) has shown promising clinical results. A case report showed stabilization (>6 months) of brain and epidural metastatic lesions in a 34-year-old patient with HER2+ BCBM treated with repeated intraventricular injections of trastuzumab. Another case report showed significant improvement in general health (approximately 11 months) in a 39-year-old patient with HER2+ breast cancer and leptomeningeal carcinomatosis after repeated intrathecal injections of trastuzumab [Bousquet, G., et al., Intrathecal Trastuzumab Halts Progression of CNS Metastases in Breast Cancer, Journal of Clinical Oncology, 2016, 34(16):e151-e154, epub June 1, 2016]. Nevertheless, the long-term feasibility of this approach is limited by the need for repeated drug administration to overcome its dramatically short half-life in the CSF versus systemic circulation (approximately 1 day versus 5 days, respectively).

[0210] Trastuzumab expression test To achieve sustained CNS expression of trastuzumab, we developed a gene therapy approach to treat HER2+ BCBM, involving a single local administration of an adeno-associated virus (AAV) vector encoding trastuzumab (engineered coding sequence, i.e., trastuzumab-coGW). Sixteen novel AAV-trastuzumab vectors were engineered to evaluate trastuzumab expression efficiency within the CNS. All vector repeats were packaged into AAV rhesus 91 (AAVrh91) capsids and injected via the intracerebroventricular (ICV) route into 7-8 week-old adult female Rag1 knockout (KO) mice (N=4-5 mice / vector group) for subsequent gene expression studies. Phosphate-buffered saline (PBS)-injected Rag1 KO mice were used as a vehicle sham control group (N=5 mice). The Rag1 KO mouse model was chosen for this purpose because it is the preferred model for tumor challenge studies to confirm HER2 targeting using human-derived xenograft tumor models. Submandibular (Sub-M) blood collection was performed on days -1, 7, 14, 21, and 28. Euthanasia was performed on day 28 via whole body perfusion with PBS.

[0211] To quantify trastuzumab expression, 1 × 10 11 A 28-day time course ELISA (enzyme-linked immunosorbent assay) analysis study was performed using serum and brain homogenates (end time point) from whole body perfused mice treated ICV with each AAVrh91-trastuzumab vector or PBS at genome copies (GC) / mouse (Table 1 below). [Table 1]

[0212] FIG. 2A shows the results of an ELISA with plotted measurements of trastuzumab concentration (ng / mg protein) in perfused brain tissue samples collected following dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40 and AAVrh91.UbC.CI.IL2_V1_trastuzumab-coGW.SV40 (Promega Intron) in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. FIG. 2B shows the results of an ELISA with plotted measurements of trastuzumab concentration (μg / mL) in serum samples collected following dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40 and AAVrh91.UbC.CI.IL2_V1_trastuzumab-coGW.SV40 (Promega Intron) in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. The mean peak serum concentration in subjects receiving 500 mg (highest dose) was 377 μg / mL. FIG. 4A shows further data measuring trastuzumab concentrations in serum samples collected 28 days after dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. FIG. 4B shows further data measuring trastuzumab concentrations in perfused brain tissue samples collected 28 days after dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS.

[0213] In addition, Figure 8A shows that ICV injection of AAV resulted in sustained expression of trastuzumab in Rag11 KO mice as measured by ELISA on serum samples, and Figure 8B shows that ICV injection of AAV resulted in sustained expression of trastuzumab in Rag11 KO mice as measured by ELISA on brain homogenate (perfused) samples.

[0214] Additionally, vector biodistribution within the brain and liver was assessed to help determine the extent of brain and peripheral transduction following ICV administration (Table 2, below). [Table 2]

[0215] Figures 3A and 3B show summary diagrams of the data in Table 1. Figure 3A shows the results of a biodistribution study with plotted measurements of DNA concentration (GC / μg) in collected liver and brain tissues. Figure 3B shows the results of a biodistribution study with plotted measurements of RNA concentration (copies / 100 ng of RNA) in collected liver and brain tissues. Figure 4C shows further data measuring DNA distribution in collected brain and liver tissue samples 28 days after dosing with AAVrh91.CB-CI.IL2_V1_trastuzumab-coGW.SV40, AAVrh91.UbC.PI.IL2_V1_trastuzumab-coGW.SV40, and AAVrh91.UbC.PI.IL2_V2_trastuzumab-coGW.SV40 in mice compared to AAVhu68.CMV.PI.trastuzumab.SV40 (previously examined construct) and PBS. All vectors tested were well tolerated in mice and did not raise any safety concerns.

[0216] Trastuzumab was detected in mouse serum and brain by ELISA after a single ICV delivery of all 16 AAVrh91-trastuzumab vectors (Figures 2A and 2B). Among the CB promoter cohort, the trastuzumab-coGW construct encoding the human engineered trastuzumab sequence (AAVrh91.CB7.CI.IL2_V1.trastuzumab-coGW.SV40) resulted in the highest expression in brain and the lowest systemic concentration in serum 28 days after ICV delivery (average 26 ng antibody per mg protein). Western blot analysis suggested equimolar ratios of antibody heavy and light chains, suggesting correct assembly (Figures 11A and 11B). Figures 11A and 11B show representative Western blots confirming the expression of trastuzumab heavy and light chains in brain lysates (e.g., Figures 2A and 2B).

[0217] To further improve on the current engineered lead vector, CB.CI.IL2_V1.trastuzumab-coGW.SV40, a study was conducted to evaluate the transduction efficiency of CB.CI.IL2_V1.trastuzumab-coGW.SV40 packaging into AAVhu95 capsids. Briefly, in the AAVhu95 capsid evaluation study, CB.CI.IL2_V1.trastuzumab-coGW.SV40 and an isotype control vector (i.e., 3bnc117 antibody-encoding control vector) were packaged into AAVhu95 capsids and administered to mice via ICV injection. AAVhu95 transduction efficiency was assessed for trastuzumab expression using ELISA and via fluorescent imaging. Figure 5A shows expression levels of trastuzumab (μg / mL) measured in serum samples on days −1, 7, 14, and 28 after administration with AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 compared to capsid control and PBS. Figure 5B shows expression levels of trastuzumab (μg / mL) measured in brain tissue samples on days −1, 7, 14, and 28 after administration with AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 compared to capsid control and PBS. FIG. 6 shows vector biodistribution (GC / diploid cells) samples at days −1, 7, 14, and 28 following dosing with AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40, AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 compared to capsid control and PBS.

[0218] Testing of rAAV.trastuzumab-coGW (also referred to as AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40 or AAV.trastuzumab) preclinical activity used the mouse models described in Table 3 immediately below, including trastuzumab sensitivity and expected outcomes. Briefly, xenograft implantation procedures include guide screw implantation and tumor cell engraftment (>1 week later), see also Lal, et al., J Neurosurg 92:326-333, 2000. [Table 3]

[0219] Preclinical activity in preventive and therapeutic models (also referred to as disease remission models) was examined. Figure 1A shows a schematic diagram of the study designed to examine preclinical activity in mice. Briefly, in the preventive model (preventive model with BT-474 (ER+ / PR+ / HER2+) cell line), guide screws were implanted on days -35 to -28, and Rag1-KO female, approximately 7-8 weeks old mice were injected with 100 mg of erythropoietin via ICV injection on day -21 (21 days prior to cell implantation). 11Mice were treated with AAV at a dose of 1000 mg / mouse, implanted with estrogen pellets on day -1, and intracranial implantation of the BT-474 cell line on day 0. Survival was monitored until human endpoint. Figure 1B shows results demonstrating preclinical activity of engineered trastuzumab (antitumor activity against breast cancer brain metastases) shown as a plot of survival probability in mice after tumor implantation. Figure 10 shows Kaplan-Meier survival analysis of survival probability in tumor-bearing mice treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 (preventative treatment). For endpoints, signs of disease progression used included reduced mobility, hunched posture, and weight loss. Median survival in the indicated groups was determined: PBS, n=12, 7 weeks (53 days); isotype control, n=12, 5 weeks (37.5 days); AAV.trastuzumab, n=12, not reached. These results indicate that prophylactic treatment with AAVrh91.CB.CI.IL2_V1.trastuzumab-coGW.SV40 and AAVhu95.CB.CI.IL2_V1.trastuzumab-coGW.SV40 prevented the development of HER2+ brain tumors. Pretreatment with AAV.trastuzumab prevented the progression of brain xenografts with 100% survival over 10 weeks.

[0220] Additionally, preclinical activity in the BT-474 (ER+ / PR+ / HER2+) mouse model (i.e., treatment of breast cancer brain xenografts with AAV.Trastuzumab) was investigated. Figure 14A shows a schematic of the experimental design of the study investigating preclinical activity in mice. Briefly, on day -7 (i.e., 7 days prior to tumor cell implantation), guide screws were implanted, on day 0 tumor cells were implanted, and on day 3 (after tumor cell implantation), AAVhu95.CB.CI.IL2_V1.Trastuzumab-coGW.SV40 was administered at a dose of 1e11 (1x10 11GC / mouse) and then imaging was performed to monitor tumor burden until humane endpoint. FIG. 14B shows quantification of tumor burden assessment (bioluminescence assessment) in mouse xenografts (BT-474 (ER+ / PR+ / HER2+) xenografts) following treatment with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 compared to PBS and isotype controls. FIG. 14C shows Kaplan-Meier survival analysis of survival probability in tumor-bearing mice (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40. Briefly, human endpoints included signs of disease progression such as decreased mobility, hunched posture, and weight loss. Median survival times were determined in the indicated groups: PBS, n=8, 4 weeks; isotype control, n=10, 5 weeks; AAV.trastuzumab, n=14, not reached. These results indicate that treatment with AAV.trastuzumab on day +3 inhibited tumor progression.

[0221] Additionally, we investigated preclinical activity in the MDA-MB-453 (ER- / PR- / HER2+) (also referred to as MDA-453, highly sensitive to trastuzumab) mouse model. Briefly, in a disease remission model on day 0, intracranial implantation of the MDA-453 luciferase+ cell line was performed, and on day 3, AAV treatment (10 11GC / mouse) and survival to humane endpoints. Figure 7 shows quantification of tumor bioluminescence assessment in mouse xenografts (MDA-MB-453 (ER- / PR- / HER2+)) following treatment with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40 compared to isotype control (isotype control: 47 days, PBS: 44 days, rAAV.Trastuzumab-coGW: >6 weeks). Efficacy of rAAV.Trastuzumab-coGW injected after establishment of Her2+ cancer in mouse brain. Complete tumor remission was observed as measured by tumor growth (chart) and survival (inset). Representative bioluminescence images were taken at weeks 4, 5, and 6 for quantitative analysis of tumor burden assessment by IVIS (bioluminescence assessment) in mouse xenografts (MDA-MB-453(ER- / PR- / HER2+)) following treatment with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 compared to isotype control (data not shown). In addition, representative bioluminescence images were taken for quantitative analysis of tumor bioluminescence assessment in mouse xenografts (MDA-MB-453(ER- / PR- / HER2+)) following treatment with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 compared to isotype control. Additionally, Figure 9 shows a Kaplan-Meier survival analysis of survival probability (disease remission) in tumor-bearing mice treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40. Median survival times were determined for the indicated groups: PBS, 6 weeks (44 days); isotype control, 6 weeks (47 days); AAV.trastuzumab, not reached. These results show that complete disease remission was achieved in tumor-bearing mice treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 with a 100% survival rate for over 10 weeks.

[0222] Additionally, a dose comparison study was performed using the MDA-MB-453 mouse model to examine long-term transgene expression after a single (or two, re-administration on day 59 after tumor cell implantation) ICV administration of AAV.Trastuzumab. Figure 15A shows a schematic of the experimental design of the study examining long-term transgene expression after a single or two (re-administration) doses of AAV.Trastuzumab. Figure 15B shows trastuzumab expression levels measured by ELISA using serum samples taken on day 98, plotted as μg Trastuzumab / mL. Figure 15C shows trastuzumab expression levels measured by ELISA using brain samples taken on day 98 (perfused brain homogenate), plotted as μg Trastuzumab / mL. These results show that trastuzumab is stably expressed for >98 days after a single ICV injection.

[0223] Additionally, preclinical activity was examined in BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts, providing an assessment in a more rigorous model. Figure 12A shows a schematic of the experimental design of the study to examine preclinical activity in mice. Figure 12B shows quantification of tumor bioluminescence assessment in mouse xenografts (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) following treatment with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40 compared to isotype control. Figure 12C shows Kaplan-Meier survival analysis of survival probability in tumor-bearing mice (BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts) treated with AAVhu95.CB.CI.IL2.V1.trastuzumab-coGW.SV40. These results show that treatment of BT-474 clone 5 trastuzumab-resistant (ER+ / PR+ / HER2+) xenografts with AAV.trastuzumab on day +3 delayed tumor progression, with median survival times observed to be 4 weeks for isotype control, 4 weeks for vehicle control, and 7.5 weeks for AAV.trastuzumab (i.e., approximately 2-fold longer survival).

[0224] Additionally, preclinical activity in the MDA-MB-231-HER2 / low tumor model was investigated. Figure 13A shows a schematic of the experimental design of the study to investigate preclinical activity in mice. Figure 13B shows a Kaplan-Meier survival analysis of survival probability in tumor-bearing mice (MDA-MB-231HER2 / low tumor xenografts) treated with AAVhu95.CB.CI.IL2.V1.Trastuzumab-coGW.SV40. Figure 13C shows Her2 expression levels in MDA-MB-231 cells measured via flow cytometry after surface staining with an isotype control antibody. Figure 14D shows Her2 expression levels in MDA-MB-231 cells measured via flow cytometry after surface staining with a Her2 antibody. These results show that treatment with AAV.Trastuzumab ICV on day +3 inhibited the progression of MDA-MB-231HER2 / low tumors.

[0225] Overall, single-dose ICV administration of AAV.CB.CI.IL2_V1.trastuzumab-coGW.SV40 resulted in robust anti-tumor responses in the HER2+ breast-to-brain metastasis xenograft mouse models tested.

[0226] Treatment with AAV-trastuzumab vectors driven by CB or UbC promoters resulted in sustained antibody expression after 28 days. This gene therapy approach has the potential to provide and maintain long-lasting steady-state levels of drug in the brain after a single treatment of localized delivery of trastuzumab to prevent and / or treat HER2+ metastatic brain lesions after a single administration, as well as an effective one-time gene therapy approach for the prevention and / or treatment of HER2+ BCBM. This therapeutic strategy for HER2+ BCBM uses different combinations of HER2 modulators (anti-HER2 monoclonal antibodies and kinase inhibitors) and chemotherapeutic agents. Furthermore, intracranial delivery of AAV-trastuzumab in HER2+ breast cancer patients at high risk of developing BCBM can prevent or significantly delay the appearance of tumor lesions. CNS delivery of AAV-trastuzumab may be used as an alternative approach to achieve disease remission or stabilization, either alone or in combination with standard of care systemic HER2 kinase inhibitors, as it circumvents the therapeutic landscape and may result in long-lasting trastuzumab expression.

[0227] Natural History Tumor Progression Study This study evaluates the validity of selected tumor model approaches. Briefly, in this study, green fluorescent protein positive (GFP+) breast cancer cells (e.g., BT-474, BT-474 clone 5 (in vivo trastuzumab resistant and aggressive cell line), MDA-MB-453, MDA-MB-231 cells) were selected and expanded after selection. Cells were then seeded at 1.5 × 10 in PBS / Matrigel (50:50) using the guide screw method. 5The tumor cells were then implanted in the tumor-cell-engrafted foci (e.g., guide screw implantation followed by >1 week post-tumor cell engraftment). Advantages of the guide screw method are that it does not require a stereotactic frame, is streamlined (i.e., more animals per treatment (~6 min / mouse)), the screw can be implanted even weeks prior to tumor cell implantation, and allows intratumoral delivery of therapeutic agents (i.e., the screw can be restarted several times). Hematoxylin and eosin (H&E) histology has thus far demonstrated 100% tumor acquisition (data not shown). Additionally, we have generated a GFP / luciferase dual reporter breast cancer cell line that is also used in the studies herein.

[0228] Additionally, patient-derived xenograft models were developed using HER2+ breast cancer fragments from (DFCI) that were further implanted into tumor slurry in 6-8 week old NODscid gamma mice (NSG™ mice, Jackson Laboratory) and had a time to progression of 6-12 months.

[0229] Furthermore, the studies in mice will be expanded to include tumor challenge experiments using HER2+ and HER2- xenograft mouse models to confirm antitumor efficacy and target specificity, as well as combination therapy approaches including clinical HER2 kinase inhibitor(s) to maximize antitumor efficacy. Further studies will include evaluation of AAV-trastuzumab vectors packaged in AAVhu95 capsids. The AAV-trastuzumab vectors will then be further evaluated for toxicity in rhesus monkeys.

[0230] Example 3: Further evaluation of rAAV.trastuzumab in mice First, expression levels in mice of modified rAAV.Trastuzumab vectors were evaluated, including vectors that contain a Furin cleavage site in the linker sequence (Furin / T2A) between the heavy and light chains of the Trastuzumab coding sequence: AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Furin_V1_Trastuzumab-coGW.SV40.

[0231] Figures 16 and 17 show a comparison of the assessment of expression levels of trastuzumab in mice following administration of rAAV.trastuzumab and modified rAAV.trastuzumab. Figure 16 shows the expression levels of trastuzumab in serum following administration of AAVhu95M199.CB7.CI.IL2_trastuzumab-coGW.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1_trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_trastuzumab-coGW.SV40 in mice. FIG. 17 shows trastuzumab expression levels in the brain (perfused) following administration of AAVhu95M199.CB7.CI.IL2_trastuzumab-coGW.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1_trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1_trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_trastuzumab-coGW.SV40 in mice.

[0232] Next, a study was conducted to determine the lowest effective dose to confirm the therapeutic effect of rAAV.trastuzumab. Figure 18A shows a schematic diagram of the experimental design of the study to evaluate tumor burden. Briefly, guide screws were implanted 7 days before cell implantation (D-7), and MDA-MB-453 cells were implanted on day 0 (2.5 × 10 5cells / mouse), and 3 days after cell transplantation, mice were injected with 1 × 10 11 GC / mouse were administered intracranially (ICV) and imaged at 2, 4, and 6 weeks after cell implantation. Figure 18B shows the results of tumor burden assessment plotted as total flux (p / s) at 2, 4, and 6 weeks after tumor cell implantation in mice treated with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40, and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. These results confirm the therapeutic efficacy of the rAAV.Trastuzumab vector. FIG. 18C shows Kaplan-Meier survival curves in mice following treatment with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40.

[0233] Next, AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40 were administered at a dose of 1 × 10 10 GC / mouse and 1×10 11 Tumor challenge and tumor remission studies were performed in MDA-MB-453 xenograft mouse models administered GC / mouse. Figure 21A shows a schematic of the experimental design of the study to evaluate tumor challenge. Briefly, guide screws were implanted 7 days prior to cell implantation (D-7) and MDA-MB-453 cells were implanted on day 0 (2.5×10 5 cells / mouse), and 3 days after cell transplantation, mice were injected with 1 × 10 11GC / mouse at a dose of 1x10 and imaging was performed at 2, 4, and 5 weeks after cell transplantation. 11 Figure 22A shows tumor growth plotted as total flux (p / s) measured at weeks 2, 4, and 5 after tumor implantation in mice treated with a dose of GC / mouse. Figure 22B shows a schematic of the experimental design of the study to evaluate tumor challenge. Briefly, guide screws were implanted 7 days prior to cell implantation (D-7) and MDA-MB-453 cells were implanted on day 0 (2.5×10 5 cells / mouse), and 3 days after cell transplantation, mice were injected with 1 × 10 10 GC / mouse at a dose of 1×10 and imaged at 2, 4, and 5 weeks after cell transplantation. 10 Figure 23A shows tumor growth plotted as total flux (p / s) measured at weeks 2, 4, and 5 after tumor implantation in mice treated with 1x10 GC / mouse doses of AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. 10 GC / mouse and 1×10 11 Figure 23B shows the results of tumor burden assessment examined by imaging plotted as total flux (p / s) in mice treated with GC / mouse. 10 GC / mouse and 1×10 11 Shown are the results of Kaplan-Meier survival analysis plotted as survival probability in mice treated with GC / mouse.

[0234] Additionally, a study was conducted to evaluate antitumor activity in a BT474 clone 5 cell xenograft tumor model. Figure 19A shows a schematic diagram of the experimental design of the study to evaluate antitumor activity. Briefly, guide screws were implanted 7 days before cell implantation (D-7), and E2 pellets with BT474 clone 5 cells were implanted on day 0 (1.5 x 10 5 cells / mouse), and 3 days after cell transplantation, mice were injected with 1 × 10 11 GC / mouse were administered intracranially (ICV) at a dose of 100 mg / mL and imaged 2 weeks after cell implantation. Figure 19B shows the results of tumor burden assessment plotted as total flux (p / s) at 2, 4, and 6 weeks after tumor cell implantation in mice treated with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. FIG. 19C shows Kaplan-Meier survival curves in mice (bearing BT474 xenografts) following treatment with AAVhu95M199.UbC.CI.IL2.3bnc117.SV40, AAVhu95M199.IE.CB.CI.3bnc117.SV40, AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.trastuzumab-coGW.SV40, AAVhu95M199.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40.

[0235] Next, rAAV vector titer testing was performed to examine trastuzumab expression levels. Detection and quantification of trastuzumab was performed by mass spectrometry (LC-MS) and ELISA in perfused mouse brain homogenates. Figure 20 shows that rAAV.trastuzumab (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40) was injected at 1 × 10 11 GC / mouse and 1×10 10The results of the vector titer study plotted as trastuzumab expression levels measured by ELISA and mass spectrometry in mice administered a dose of GC / mouse. These results were obtained using 1x10 10 We show that the dose of GC / mouse is sufficient to produce transgene levels similar to those seen in NHP brains and induce sustained tumor remission in MDA-MB-453 brain xenografts.Low doses of rAAV.trastuzumab inhibit tumor progression in a xenograft model derived from a HER2 cell line.

[0236] Additionally, a study was conducted to examine the effect of IVIG pretreatment in mice followed by ICV administration of rAAV.Trastuzumab (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). Briefly, healthy TAG1KO female mice were inoculated with 1 × 10 11 rAAV.Trastuzumab was administered via ICV at a dose of 100 mg / mouse and blood samples for analysis were taken 3 days prior to rAAV administration and 7, 14, 21, and 29 days after administration. Figure 24A shows trastuzumab expression levels in serum after administration of rAAV.Trastuzumab with or without IVIG pretreatment. Figure 24B shows trastuzumab expression levels in the brain (perfused) at day 30 after administration of rAAV.Trastuzumab with or without IVIG pretreatment. These results indicate that pretreatment with IVIG reduced systemic levels of trastuzumab but not in the brain.

[0237] Overall, the above results demonstrate that the engineered trastuzumab constructs in AAV.trastuzumab were able to induce 1×10 11 These data confirm that a dose of 1×10 GC / mouse inhibited tumor progression. Additionally, these data support the use of lower doses (i.e., 1×10 10 GC / mouse) was still sufficient to induce tumor remission in MDA-MB-453 xenografts (HR- / HER2+).

[0238] Example 4: rAAV Evaluation in Non-Human Primates (NHP) This study investigated rAAV.trastuzumab-coGW vector efficacy in non-human primates (NHPs, Nab+ female Indian rhesus macaques). Briefly, baseline measurements of clinical pathology samples were performed on day -5, as well as neutralizing antibody (NAb) levels, neurological examination, and nerve conduction velocity (NCV) testing. On day 0, NHPs were administered specific AAV vectors (via intracisternal (ICM) injection), at which time additional clinical pathology samples were taken, serum and cerebrospinal fluid (CSF) samples were collected for analysis, and NAbs were assessed. On days 7 and 21, additional serum and cerebrospinal fluid (CSF) samples were collected for analysis. On day 14, additional serum and cerebrospinal fluid (CSF) samples were collected for analysis, and additional neurological examination was performed. On days 36-37 after ICM, necropsies were performed, further clinical pathology samples were taken, serum and CSF fluid samples were collected, NAb / PBMC were assessed, and further neurological and NCV testing was performed prior to necropsy. ELISA and humoral immune responses (anti-drug antibodies (ADA)) for trastuzumab were performed throughout the study to assess development and further into the study period. Table 3 below shows a schematic summary of the study design. [Table 4]

[0239] Figure 25A shows trastuzumab expression levels in CSF measured by ELISA at days 0.3, 7, 14, 21, and 36-37 after rAAV administration in NHPs from cohort 1b ((AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40)), cohort 2 (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40), and day 14 in the negative control. FIG. 25B shows levels of anti-drug antibodies (ADA) measured in CSF samples collected in NHPs on days 0.3, 7, 14, 21, and 36-37 after rAAV administration (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). These results show that trastuzumab is detectable in the CSF up to the final time point tested and ADA to trastuzumab is not detectable in the CSF.

[0240] Figure 26A shows trastuzumab expression levels in serum measured by ELISA at days 0.3, 7, 14, 21, and 36-37 after rAAV administration in NHPs from cohort 1b ((AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40)) and cohort 2 (AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). FIG. 26B shows levels of anti-drug antibodies measured in serum samples collected in NHPs on days 0, 3, 7, 14, 21, and 36-37 after rAAV administration (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40). These results show that after ICM treatment with AAV.Trastuzumab, there is low systemic trastuzumab detected in NHP serum by ELISA.

[0241] Figure 27 shows quantification of trastuzumab protein in NHP brain tissue following ICM administration of AAV.trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40). Figure 28A shows quantification of trastuzumab protein in NHP spinal cord following ICM administration of AAV.trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40). FIG. 28B shows quantification of trastuzumab protein in NHP dorsal root ganglia (DRG) following ICM administration of AAV.trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40).

[0242] Figures 29A-29D show the results of 10XGENOMICS (final sequencing depth average number of reads - 883 × 10 6Figure 29 shows the results of trastuzumab mRNA detection in NHP brain tissues (cerebellum and occipital cortex) by 100% seq. reads / sample. No background detection of trastuzumab was observed in untreated monkeys. Figure 29A shows the results of trastuzumab mRNA detection in the cerebellum in NHP18-032 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40. Figure 29B shows the results of trastuzumab mRNA detection in the cerebellum in NHP20-198 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.trastuzumab-coGW.SV40. Figure 29C shows the results of trastuzumab mRNA detection in the occipital cortex of NHP18-032 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40. Figure 29D shows the results of trastuzumab mRNA detection in the occipital cortex of NHP20-198 administered AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40.

[0243] Figures 30A and 30B show the biodistribution of AAV.Trastuzumab (AAVhu95M199.IE.CB7.CI.IL2_Furin_V1.Trastuzumab-coGW.SV40 and AAVhu95M199.UbC.PI.IL2_Furin_V1.Trastuzumab-coGW.SV40) in NHPs in various tissues after ICM administration. Figure 30A shows the DNA biodistribution of AAV.Trastuzumab in NHPs in various tissues after ICM administration. Figure 30B shows the RNA biodistribution of AAV.Trastuzumab in NHPs in various tissues after ICM administration.

[0244] Overall, these results indicate that treatment with AAV.trastuzumab can both prevent the development of HER2+ CNS tumors and drive tumor regression in the tumor animal models evaluated to date.

[0245] Example 5: Results of adeno-associated virus (AAV) vector-mediated delivery of trastuzumab to the central nervous system in a Her2+ breast cancer brain metastasis and non-human primate xenograft model Translation Relevance The incidence of breast cancer metastasis to the central nervous system (CNS) is increasing due to advances in systemic therapies that better control extracranial disease but potentially allow metastatic cells within the CNS to escape latency. Breast cancer patients with symptomatic brain and / or leptomeningeal metastases have a poor prognosis because current adjuvant therapies cannot prevent CNS recurrence. Repeated intrathecal (IT) delivery of trastuzumab is safe and provides clinical benefit to patients with HER2+ CNS disease. Trastuzumab (Herceptin®) has been approved in protein form as the gold standard treatment for human epidermal growth factor receptor 2 (HER2)-positive breast cancer. To overcome the treatment burden associated with continuous IT drug administration, we developed an adeno-associated virus (AAV) vector that persistently expresses trastuzumab within the CNS after a single IT injection. Preclinical studies have demonstrated that AAV-trastuzumab induced remissions in a HER2+ orthotopic model of brain metastases and was well tolerated in non-human primates. These encouraging preclinical findings justify future clinical trials.

[0246] Abstract Single-dose gene therapy expressing trastuzumab-like anti-Her antibodies within the CNS represents a therapeutic option for disease prevention or treatment.

[0247] For IT administration, we developed an AAV vector expressing an engineered trastuzumab sequence driven by the ubiquitin C (UbC) promoter (AAV9.UbC.Trastuzumab). CNS transgene expression was assessed after a single intracerebroventricular (ICV) or intracisternal (ICM) AAV9.UbC.Trastuzumab injection in adult Rag1 knockout mice and rhesus non-human primates (NHPs), respectively. Real-time PCR, ELISA, Western blot, in situ hybridization, immunohistochemistry, single nucleus RNA sequencing, and liquid chromatography-mass spectrometry were used. Efficacy of AAV9.UbC.Trastuzumab was determined using brain xenografts derived from HER2+ breast cancer cell lines (BT-474 and MDA-MB-453).

[0248] Transgene expression was determined using AAV9.UbC.trastuzumab (1 × 10 11 AAV9.UbC.Trastuzumab was detected in brain homogenates and serum of Rag1 knockout mice following ICV injection of 1000 vector genome copies (GC) / mouse, with levels plateauing 28 days after dosing. A single AAV9.UbC.Trastuzumab dose inhibited tumor progression in the xenograft models tested compared to AAV9.UbC.isotype control. In NHPs, AAV9.UbC.Trastuzumab (3 × 10 13 ICM delivery of GCs / animal) was well tolerated and resulted in detectable transgene expression in CNS tissues and cerebrospinal fluid after 36-37 days.

[0249] These results demonstrate that AAV9.UbC.trastuzumab showed robust activity against HER2+ brain orthotopic xenografts. With the proven clinical safety record of AAV9, this gene therapy may represent a viable approach for targeting HER2+ CNS malignancies.

[0250] To fill the need for novel therapies targeting HER2+ CNS metastases, we developed a gene therapy approach involving a recombinant adeno-associated virus (AAV) vector engineered to constitutively express trastuzumab within the CNS. Effective single-dose gene therapy provides a continuous local source of trastuzumab, improving patient outcomes.

[0251] Here, we demonstrate that a single IT administration of AAV9 encoding engineered trastuzumab driven by the human ubiquitin C (UbC) promoter (AAV9.UbC.Trastuzumab) resulted in a robust antitumor response in a treatment setting against a HER2+ xenograft BCBM model. We also show that intracisternal injection of AAV9.UbC.Trastuzumab in rhesus NHPs was well tolerated and resulted in detectable transgene protein expression at levels expected to exert a therapeutic effect against an orthotopic cell line-derived xenograft BCBM mouse model. Overall, AAV-mediated expression of trastuzumab within the CNS may represent a viable strategy to treat and prevent the emergence of HER2+ CNS metastases in breast cancer patients.

[0252] Materials and Methods Vector construction Transgene expression cassettes containing engineered trastuzumab coding sequences were synthesized by GeneArt (ThermoFisher) and engineered as follows: the heavy and light chains were preceded by a human interleukin 2 (IL2) signal peptide (MYRMQLLSCIALSLALVTNS), separated by a furin cleavage site, followed by a T2A self-cleaving peptide linker and a mutant IL2 signal peptide (MYRMQLLLLIALSLALVTNS). The UbC promoter was used to drive transgene expression (Figure 31A).

[0253] AAV9. Isotype vector controls were engineered to express 3BNC117, a fully human IgG1 kappa monoclonal antibody against the HIV envelope. All vectors were packaged into AAV9 capsids and produced and purified by the Penn Vector Core as previously reported [Lock, M., et al., Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Hum Gene Ther, 2010. 21(10): p. 1259-71].

[0254] Generation of Cell Lines and GFP-Luciferase+ Breast Cancer Reporter Cell Lines Human breast cancer cell lines MDA-MB-453 (HTB-131) and BT474 (HTB-20) were obtained from ATCC and maintained according to the vendor's instructions.

[0255] Cell lines were transduced with the CMV-luciferase-EF1α-copGFP BLIV2.0 lentivector lentiviral vector using TransDux MAX lentiviral transduction reagent according to the vendor's instructions (Systems Biosciences). Transduced cells were then sorted for the top 50% brightest cells expressing green fluorescent protein (GFP).

[0256] Animal procedures All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Animals were visually monitored daily by animal care and / or veterinary staff for any signs of distress requiring intervention.

[0257] Vector administration Mice: AAV vectors were administered intracerebroventricularly (ICV) to 7-8 week-old female B6.129S7-Rag1tm1Mom / J knockout mice (Rag1 KO, Jackson Laboratories strain no. 002216) by trained laboratory animal technicians or veterinarians (success rate >95%). Blood was collected from the submandibular vein at the planned time points (Figure 31B).

[0258] Non-human primates (NHP): Adult female Indian rhesus macaques (3-4 years old) were administered 1 mL of AAV vectors via intracisternal delivery (ICM) as previously reported [Katz, N., et al., Standardized Method for Intra-Cisterna Magna Delivery Under Fluoroscopic Guidance in Nonhuman Primates. Hum Gene Ther Methods, 2018. 29(5): p. 212-219]. Serum, CSF, and tissue samples from Indian rhesus macaques previously ICM-treated with AAV9.GFP were used as negative controls in the transgene expression assay. Blood was collected from a peripheral vein, and up to 1 mL of CSF was collected via suboccipital puncture (Figure 34A). Animals were clinically monitored by veterinarians throughout the study, and the main parameters assessed were vital signs, clinical pathology of blood and CSF, and comprehensive neurological evaluation (mental status, posture, proprioception, gait, reflexes, nerve conduction velocity testing).

[0259] autopsy Mice: For vector expression studies, animals were deeply anesthetized and systemically perfused with saline through the left ventricle at the planned study endpoint. Brain, heart, and liver tissues were snap frozen for analysis.

[0260] NHP: 36-37 days after vector administration, animals were deeply anesthetized and euthanized by intravenous (i.v.) pentobarbital overdose, followed by cerebral perfusion by injecting 600 mL of saline into the jugular vein. Various tissues were collected for histopathological analysis and tissues collected included: adrenal gland, aorta, bone marrow, femur, bone, sternum, brain: right hemisphere, brain: caudate nucleus, brain: cerebellum, brain: frontal cortex, brain: hippocampus, brain: medulla, brain: occipital cortex, brain: parietal cortex, brain: temporal cortex, brain: thalamus, brain: olfactory bulb, chest: tatoe, deep cervical lymph nodes, cervical dorsal root ganglion, thoracic dorsal root ganglion, lumbar dorsal root ganglion, sacral dorsal root ganglion, esophagus, eye, gallbladder, heart, large intestine, colon, small intestine, duodenum, small intestine, jejunum, kidney (left), kidney (right), liver: left lobe, liver: right lobe, liver: middle lobe, liver: caudate lobe, lung: left lobe, lung: right lobe, lymph nodes: axillary, lymph nodes: cervical, lymph nodes: inguinal, lymph nodes: mesenteric. Muscle:Quadriceps, Muscle:Gastrocnemius, Nerve:Optic (Cranial Nerve II), Nerve:Sciatic, Nerve:Sural, Nerve:Proximal Midline, Nerve:Distal Midline, Nerve:Median Epon, Nerve:Tibia, Ovary, Pancreas, Salivary Gland:Submandibular, Injection Site:Skin and Surrounding Tissues, Injection Site:Spinal Cord, Spinal Cord:Cervical, Spinal Cord:Thoracic, Spinal Cord:Lumbar, Spleen, Thymus, Thyroid with Parathyroid, Trachea, Trigeminal Nerve+Ganglion, Bladder, Gross Lesion.

[0261] Tumor models Breast cancer cell lines (250,000 cells / mouse) were resuspended in 1:1 DPBS / Matrigel and implanted into 7-8 week old female Rag1 KO mice via guide screws as previously reported [Lal, S., et al., An implantable guide-screw system for brain tumor studies in small animals. J Neurosurg, 2000.92(2):p.326-33]. Three to four days after engraftment, mice were randomized into treatment groups using an online randomization tool (random.org) and treated with AAV vectors via ICV injection. Tumor progression was monitored using an IVIS Spectrum in vivo imaging system (PerkinElmer). Animals were euthanized at humane endpoints (signs of cachexia, lethargy, hunched posture).

[0262] Estrogen receptor (ER), progesterone receptor (PR), and HER2 receptor tumor status was established by immunohistochemistry on formalin-fixed brain sections.

[0263] histopathology Histopathological evaluation of NHP tissues was performed by a veterinary pathologist certified by the American College of Veterinary Pathologist using formalin-fixed, paraffin-embedded tissues stained with hematoxylin and eosin.

[0264] Immunohistochemistry (IHC) Immunohistochemistry was performed on sections from formalin-fixed paraffin-embedded tissues on a Leica Bond Rx autostainer following standard IHC protocols using the Bond polymer detection system (Leica Biosystems, DS9800) and DAB as chromogen. Primary antibodies were applied using the indicated conditions with a 30 minute incubation time to detect estrogen receptor (ER), progesterone receptor (PR), and HER2. After staining, processed slides were dehydrated through ethanol and xylene and coverslipped.

[0265] Vector DNA and RNA biodistribution DNA and RNA were extracted from snap-frozen tissue samples using the QIAmp Mini Extraction Kit (Qiagen). Vector GC levels were determined by qPCR using vector-specific primers and probes.

[0266] Single-nuclear cell RNA sequencing Nuclei isolation and snRNA-seq To isolate nuclei from frozen tissue samples, we used a modified version of a previously published nuclear isolation procedure [Zhu, Y., et al., Spatiotemporal transcriptomic divergence across human and macaque brain development. Science, 2018. 362(6420); Kati J. Ernst, KO, Josephine Bageritz, Jan-Philipp Mallm, Andrea Wittmann, Kendra K. Maaβ, Svenja Leible, Michael Boutros, Stefan M. Pfister, Marc Zuckermann, David TW Jones, Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues. bioRxiv, 2020]. In these isolations, all buffers and samples were kept on ice throughout the procedure to maintain nuclear integrity. Buffers were typically made as described below, pre-chilled, and then replenished immediately prior to use to make "complete" buffer with final concentrations of 1 mM DTT, 0.8 U / μl RNase inhibitor (Protector RNase Inhibitor, Roche), and 1× protease inhibitor (Complete Mini EDTA-free, Roche). To isolate nuclei, approximately 25 mg of frozen tissue was minced with a scalpel and transferred to a pre-chilled 2 ml Dounce homogenizer containing 1 ml of cold complete lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0, and 0.1% TritonX-100). The tissue was homogenized with 10 strokes each of mortar A then B, then passed sequentially through pre-soaked 100 μM and 30 μM filters, and the filtered sample was collected in a sterile tube. The homogenizer and filter were washed with an additional 3 volumes of complete lysis buffer and collected with the filtered sample.Two volumes of sample were then layered on top of one volume of cold complete separation buffer (1.8 M sucrose, 3 mM magnesium acetate, and 10 mM Tris-HCl, pH 8.0) and spun at 21,000 × g for 45 min at 4 °C. The supernatant was then carefully removed and 100 μl of complete resuspension buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.2) was added to the tube without mixing. Samples were incubated on ice for 10-15 min, then nuclei were completely resuspended by gently pipetting up and down 20-30 times and counted using an automated cell counter (Countess3, Thermo Fisher). For submission of single nucleus transcriptomics samples, the concentration was adjusted to approximately 1 × 10, following the manufacturer's sample loading guidelines. 6 Nuclei / ml (0.7–1.2 × 10 per ml) 6 The total number of nuclei was adjusted to the range of 10000 nuclei. To identify single nucleus transcriptomes, 10x Genomics Chromium Controller and 3' Gene Expression Assay (ver.3) was used according to the manufacturer's protocol to achieve approximately 10,000 compartmentalized nuclei per sample. Nuclear compartmentalization, reverse transcription and cleanup, cDNA amplification, and library construction and cleanup were all performed as described in the manufacturer's protocol, and libraries were sequenced on an Illumina NextSeq2000. The final sequencing depth using the average reads was 883 x 106 reads / sample.

[0267] snRNA-seq analysis After sequencing, demultiplexed Fastq files were aligned against a custom reference genome consisting of the rhesus monkey reference (Mmul 10) and the complete annotated plasmid sequence used to generate the rAAV vectors and passed through the Cell Ranger counting pipeline (10x Genomics). The Cell Ranger-generated count matrix was then further analyzed within R using the package Seurat (ver.4) as reported (Hao, Y. et al. Cell 184, 3573-3587 e3529 (2021)). Each individual sample dataset was normalized using the sctransform function, and principal component analysis (PCA), uniform manifold approximation and projection (UMAP), as well as nuclear clustering, were all performed using standard functions within Seurat for each individual dataset.

[0268] antibody Manufacturer information for all antibodies is listed in the table immediately below. [Table 5]

[0269] In situ hybridization (ISH) ISH was performed on sections from formalin-fixed paraffin-embedded tissues. Sections were manually processed using the RNAscope2.5HD Detection Kit (Advanced Cell Diagnostics, ACD) according to the manufacturer's instructions. Probes consisting of the ZZ probe pair were manufactured by ACD and were designed not to detect endogenous sequences in NHP tissues. Fast Red was used as a chromogen to reveal the sites of bound probe. Sections were mounted in Fluoromount with DAPI to counterstain nuclei and imaged with a rhodamine and DAPI filter set.

[0270] Protein extraction for ELISA and Western Blot assays Snap-frozen brain tissue was homogenized with 5 mm stainless steel beads using a bead mill homogenizer in lysis buffer (20 mM Tris-HCl, pH: 8.0, 137 mM NaCl, 10% glycerol, 1% TritonX-100, 2 mM EDTA, 1× protease inhibitor EDTA-free tablet). Lysates were clarified by centrifugation and protein concentrations were determined by BCA assay.

[0271] Enzyme-linked immunosorbent assay (ELISA) Samples and reagents were diluted in DPBS for capture or in blocking buffer (5% BSA, 0.05% Tween-20, DPBS) for detection. CSF was diluted 1:5 in blocking buffer. Brain homogenates were diluted to 2mg total protein / mL in blocking buffer. ELISA assays were performed as previously reported [Maple, L., et al., Development and validation of ELISA for Herceptin detection in human serum. J Immunol Methods, 2004. 295(1-2): p. 169-82].

[0272] Trastuzumab Mouse: 2 μg / mL of human ErbB2 / Her2 Fc chimeric protein was first coated onto a high-binding 96-well plate. Trastuzumab was detected using an HRP-conjugated anti-human kappa light chain antibody.

[0273] NHP: 1 μg / mL NHP pre-adsorbed mouse anti-human IgG antibody was used for capture. Mouse pre-adsorbed HRP-conjugated goat anti-human IgG Fc antibody was used for detection.

[0274] A standard curve was generated using 0.5 μg / mL of anti-HER2-Tra-hIgG1 antibody.

[0275] Anti-drug antibodies (ADA) 1 μg / mL of anti-HER2-Tra-hIgG1 antibody was used for capture. 1 μg / mL of anti-trastuzumab antibody was used to generate a standard curve. 2 μg / mL of HRP-conjugated ErbB2 / Her2 antibody was used for detection.

[0276] Western blot Brain lysates (25 μg / lane total protein) were heat denatured in reducing LDS sample buffer, separated by SDS-PAGE, and transferred to PVDF membranes. Electroblotted membranes were blocked in 5% skim milk. Goat anti-human IgG FC antibody was used to probe for trastuzumab heavy chain or goat anti-human kappa light chain antibody to probe for trastuzumab light chain. HRP-conjugated donkey anti-goat IgG antibody was used to detect heavy and light chains. Blots were developed using a chemiluminescent substrate.

[0277] Liquid Chromatography-Mass Spectrometry (LC-MS) Detection of trastuzumab by LC-MS in mouse and NHP brain samples was also performed by LC-MS.

[0278] LC-MS analysis Mouse brain lysate Signature peptides for LCMS analysis and multiple reaction monitoring (MRM) transitions were selected from previous literature [Russo, R., et al., Ultra-performance liquid chromatography / multiple reaction monitoring mass spectrometry quantification of trastuzumab in human serum by selective monitoring of a specific peptide marker from the antibody complementarity-determining regions. Rapid Communications in Mass Spectrometry, 2017.31(14):p.1184-1192]. Tissue samples were homogenized in lysis buffer (50 mM ammonium bicarbonate, 6 M urea, 0.15% sodium deoxycholate) at a ratio of 7 μL of buffer to approximately 1 mg of tissue. The homogenate was clarified and the protein concentration of the clarified lysate was determined by BCA. A trastuzumab calibration curve ranging from 68 to 7277 ng / mL was generated in clarified lysates from control mice. 20 μL of calibration standards, animal samples, clarified lysate blanks, and clarified lysate duplicate blanks were added in duplicate to a 1 mL 96-well plate. 50:50 methanol:acetone was added to each well, and the plate was vortexed and dried in an Eppendorf Vaccufuge without heating. Samples were reconstituted in 6 M urea and reduced in 8 mM 2-carboxyethyl)phosphine at 37° C. for 1 hour with 500 rpm shaking, followed by alkylation in 30 mM iodoacetamide at room temperature for 30 minutes in the dark. Standards, samples, and blanks were spiked with 20 μL of 10 nM heavy-labeled internal peptide standard solution (DTYIHWVR, SEQ ID NO: 56). Samples were diluted 1:3 with 0.1 mg / mL trypsin prepared in 50 mM ammonium bicarbonate. Samples were digested overnight at 37° C. with 500 rpm shaking. The digestion was quenched with 0.7% formic acid to a final concentration. Solid phase extraction was performed using Waters Oasis HLB sorbent.Eluted peptides were dried in an Eppendorf Vaccufuge without the use of heat and reconstituted in 5:95 acetonitrile:water and 0.1% formic acid. LCMS analysis was performed using an Agilent 1260 Infinity II HPLC and an Agilent TQ6495 mass spectrometer. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The stationary phase was a Waters Premier HSS T3 1.8 μm 2.1×100 mm using a gradient of 15% mobile phase B to 21% mobile phase B over 8 min. Data were analyzed and quantified using Skyline by ratio and 1 / x×x regression weighting for multiple normalization methods [MacLean, B., et al., Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics, 2010. 26(7): p. 966-968]. Calculated concentrations were normalized to BCA protein content.

[0279] NHP brain lysates Trastuzumab pull-down: Human HER2-conjugated magnetic beads were reconstituted and washed as recommended by the vendor (Acro Biosystems MBS-K006). 100 μL of beads per sample were pelleted in a microcentrifuge tube using a magnetic separator and the supernatant was discarded. Protein lysate samples from NHP brain homogenates were prepared as described in the LC-MS analysis of mouse brain lysates section. Samples were diluted to 2 mg / mL in PBS-T and 1 ml of diluted lysate was added to the tube with pelleted beads. Samples were incubated overnight at 4°C on a tube rotator. After incubation, the beads were pelleted using a magnetic separator and the supernatant was discarded. The beads were washed with PBS-T by vortexing the tube for 20 seconds, pelleting the beads on the magnetic separator and discarding the supernatant a total of four times.

[0280] Protein-bound beads were prepared according to the SP3 protocol [Hughes, CS, et al., Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nature Protocols, 2019.14(1):p.68-85]. A heavy-labeled internal standard peptide (DTYIHWVR, SEQ ID NO:56) was spiked into the solution for retention time referencing and normalization. Each sample was split into two vials for targeted and non-targeted LCMS analysis.

[0281] Vial 1 was analyzed using the targeted MRM acquisition method as discussed in the LC-MS analysis of mouse brain lysates section. Data was analyzed and quantified using Skyline by ratio versus multiple normalization methods and linear by zero single point calibration regression using a 10 ng / mL positive control as a reference [MacLean, B., et al., Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics, 2010. 26(7): p. 966-968].

[0282] Vial 2 was analyzed using a Thermo UltiMate 3000 RSLNano LC system and a Thermo Q-Exactive HF Orbitrap mass spectrometer. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The stationary phase was an Easy-Spray PepMap Neo 2 μm C18 75 μm × 150 mm using a gradient of 4% mobile phase B to 50% mobile phase B over 65 min, followed by a high organic wash and re-equilibration. Data acquisition was performed using a top 10 data-dependent acquisition method. MS1 resolution was set to 60,000 and the AGC target was set to 1e6 with a maximum IT of 60 ms scanning from 300 to 1500 m / z and an isolation window of 2 m / z. MS2 resolution was set to 30,000, AGC target was set to 1e6, with a maximum IT of 120 ms scanning from 50 to 2000 m / z, and an NCE of 27. Minimum intensity threshold was set to 8.3e3. Charge state exclusion was set to none, 1, and >7, and dynamic exclusion was set to 5 seconds. Data were analyzed using Skyline with peptide settings set to a maximum of one tryptic lacking cleavage site and fixed carbamidomethyl cysteine. Skyline transition settings were set to 2-4 ion states, ion charge of 1, and ion types of y, b, and p. Skyline product ions were set to ion 1 to last ion. Peptide mapping coverage was manually reviewed for accuracy. Relative quantification of heavy to light chains was calculated using the average MS1 peak area from the three most abundant peptides in each chain.

[0283] statistical analysis Differences in trastuzumab expression levels between treatment and control groups were analyzed using an unpaired two-tailed Student's t-test in GraphPad Prism 9.5.1. Comparisons of the number of viral genome copies between different treatment groups were calculated using t-tests on log-transformed values. Kaplan-Meier survival probabilities estimated for tumor challenge experiments in mice were plotted using GraphPad Prism 9.5.1, and differences between groups were compared using the log-rank test.

[0284] result ICV delivery of the AAV9.UbC.trastuzumab vector resulted in transgene expression in Rag1 KO mice.

[0285] To achieve stable trastuzumab expression in vivo, healthy young adult Rag1 KO female mice were injected ICV with AAV9.UbC.trastuzumab (1 × 10 11 GC / mouse) and trastuzumab levels were quantified in the systemic circulation and brain parenchyma (Figures 31A and 31B). 11ICV delivery of AAV-trastuzumab (AAV-T31C / mouse) resulted in robust systemic and local transgene expression after 4 weeks (100-1000-fold increase in signal over background, Figures 31C and 31D). Biodistribution studies in brain and liver tissues confirmed that vector delivery (Figure 31E) and transgene expression (Figure 31F) were successful in AAV-treated mice (AAV genome copies per microgram of DNA: 600-3,000-fold increase in signal over background, transgene RNA transcripts: 5,000-42,000-fold increase in signal over background). Western blot analysis confirmed expression of human IgG antibody heavy and light chains in whole brain lysates from mice that received AAV-trastuzumab via ICV injection (Figure 31G). The identity of the expressed transgene was confirmed to be trastuzumab by LC-MS with multiple reaction monitoring transitions via quantification of a unique peptide (DTYIHWVR, sequence number 56) present in brain tissue homogenates of ICV-treated mice that received AAV9.UbC.trastuzumab (signal >= 588-fold above background compared to PBS control, Figure 31H).

[0286] Figures 31A-31H show that a single ICV administration of an AAV9 vector encoding an engineered version of trastuzumab resulted in robust transgene expression in RAG1 KO mice. Figure 31A shows a schematic of the AAV vector genome. Figure 31B shows the administration of an AAV9 vector encoding trastuzumab (1x10 11 Figure 31C shows a schematic diagram for the evaluation of transgene expression in vivo following ICV delivery of AAV9.UbC.trastuzumab vector (1 x 10 11 Figure 31D shows quantification of trastuzumab by ELISA over time in serum from Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab vector (1x10 GC / mouse, n=10) or PBS (n=10). 11Figure 34 shows quantification of trastuzumab over time by ELISA in perfused brain tissue from Rag1 KO mice treated ICV with AAV9.UbC.Trastuzumab (n=4) or PBS (n=10). Figure 34E shows DNA biodistribution and Figure 31F shows RNA biodistribution analysis by qPCR in brain and liver tissue from Rag1 KO mice treated ICV with AAV9.UbC.Trastuzumab or PBS. Figure 31G shows Western blot analysis of trastuzumab heavy and light chains in brain lysates from Rag1 KO mice treated ICV with AAV9.UbC.Trastuzumab (n=4) or PBS (n=2). Purified trastuzumab (10 ng / lane) and β-actin were used as positive and loading controls, respectively. Figure 31H shows that trastuzumab identity was confirmed by LC-MS using brain homogenates from Rag1 KO mice treated ICV with AAV9.UbC.trastuzumab (n=4) or PBS (n=2). Data shown as individual data points and mean±SEM.

[0287] Preventing off-target transduction does not affect trastuzumab expression in the brains of Rag1 KO mice.

[0288] Cardiotoxicity is a prominent but largely reversible complication of systemic trastuzumab treatment observed in a subset of patients with metastatic HER2+ breast cancer. The incidence of trastuzumab-associated cardiotoxicity varies significantly with risk factors, such as pre-existing cardiac dysfunction, and the likelihood of developing any type of cardiotoxicity peaks 11 months after initiating treatment and decreases over time [Bouwer, NI, et al., Cardiotoxicity during long-term trastuzumab use in patients with HER2-positive metastatic breast cancer: who needs cardiac monitoring? Breast Cancer Res Treat, 2021.186(3):p.851-862]. Because AAV vectors may leak from the CSF into the systemic circulation after IT delivery and transduce off-target organs, we evaluated whether pooled human immunoglobulins derived from intravenous plasma (IVIg, a source of anti-AAV9 neutralizing antibodies) could reduce extracranial organ transduction after ICV AAV vector delivery in mice [Boutin, S., et al., Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther, 2010. 21(6): p. 704-12]. Compared to a control group that received naive serum, AAV9.UbC.Trastuzumab (1 × 10 11Mice that received IVIg 2 hours prior to ICV injection of GC / mouse showed a 40-fold reduction in serum trastuzumab concentrations (Figure 32A), while levels in the brain remained unaffected (Figure 32B). Vector biodistribution analysis confirmed these results (Figure 32C), indicating that reduced trastuzumab at systemic but not brain levels resulted from effective blockade of only liver and heart transduction after IVIg pretreatment. In humans, pre-existing immunity to recombinant AAV vectors after natural exposure to wild-type AAV can greatly reduce tissue transduction. However, CSF contains only a small proportion of NAbs compared to blood [Horiuchi, M., et al., Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors. Mol Ther Methods Clin Dev, 2022.27:p.272-280, Verdera, HC, K. Kuranda, and F. Mingozzi, AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Mol Ther, 2020.28(3):p.723-746], and pre-existing immunity to AAV is likely not a contraindication to this gene therapy approach for most potential patients.

[0289] Figures 32A-32C show that AAV9.UbC.Trastuzumab CNS transduction efficiency is not affected by pretreatment with intravenous immunoglobulin (IVIg) containing broadly neutralizing antibodies against AAV9. Adult female Rag1 KO mice were pretreated with AAV9.UbC.Trastuzumab (1 x 10 11Mice were pretreated with IVIg or control serum from C57BL6 / J donor mice 2 hours prior to ICV administration of GC / mice, n=5. Mice ICV-treated with PBS (n=3) were used as controls. Figure 32A shows trastuzumab levels in serum over time and Figure 32B shows trastuzumab levels in perfused brain tissue (day 30) from ICV-treated mice measured by ELISA. Figure 32C shows DNA biodistribution analysis by qPCR of AAV9.UbC.Trastuzumab vector in brain, liver, and heart 30 days after vector administration. Data shown as individual data points and mean ± SEM. ***p≦0.0001; ns, not significant difference.

[0290] Treatment with AAV9.UbC.trastuzumab inhibited tumor progression in an orthotopic xenograft mouse model of HER2+ breast cancer brain metastases

[0291] We next sought to evaluate the antitumor efficacy of AAV9.UbC.trastuzumab ICV administration in an immunodeficient mouse model that retains NK cell-mediated ADCC activity. Orthotopic xenograft Rag1 KO mouse models were generated by engrafting GFP / luciferase-reporter HER2+ cell lines (BT-474 or MDA-MB-453) in the right frontal lobe using a guide screw system [Lal, S., et al., An implantable guide-screw system for brain tumor studies in small animals. J Neurosurg, 2000.92(2):p.326-33]. The hormone receptor and HER2 status in each cell line was confirmed by IHC staining of tumor-bearing brain sections from Rag1 KO mice (data not shown). Three days after tumor cell implantation, animals were randomized into treatment groups and received either AAV9.UbC.trastuzumab or AAV9.UbC.isotype control vector (1 × 10 11 GC / mouse) by ICV injection, and tumor growth was monitored by bioluminescence imaging (data not shown).

[0292] Compared to AAV9.UbC.isotype control treated mice, AAV9.UbC.trastuzumab administration delayed tumor progression (Figure 33A) and extended OS (Figure 33B) in BT-474 tumor-bearing mice (3.8 vs. 9.9 weeks, respectively). Six of 12 mice treated with AAV9.UbC.trastuzumab remained asymptomatic for >12 weeks and were considered to be in complete remission. ICV treatment of Rag1 KO mice bearing MDA-MB-453 xenograft tumors with AAV9.UbC.trastuzumab achieved a 100% response rate (Figure 33C), with all animals remaining in remission >12 weeks after treatment, and OS for mice treated ICV with AAV9.UbC.isotype control vector was 5.5 weeks (Figure 33D). Analysis of brain homogenates from Rag1 KO mice orthotopically implanted with MDA-MB-453 104 days after ICV administration of AAV9.UbC.Trastuzumab demonstrated sustained trastuzumab expression (range 23.7-49.5 ng trastuzumab / mg total protein, Figure 33E). Together, these results provide proof-of-principle evidence supporting the use of direct CNS delivery of AAV9.UbC.Trastuzumab vectors to target HER2+ BCBM.

[0293] Figures 33A-33E show that a single dose ICV administration of AAV9.UbC.Trastuzumab resulted in tumor regression in a HER2+ breast brain metastasis xenograft mouse model. Adult female Rag1 KO mice were administered 1x10 11GC / animals were injected ICV with AAV9.UbC.Trastuzumab or AAV9.Isotype control. Figure 33A shows quantification of total photon flux via bioluminescence in vivo imaging system (IVIS), and Figure 33B shows Kaplan-Meier survival curves of mice bearing orthotopic brain tumors derived from BT-474 cell line and treated ICV with AAV vectors (AAV9.Isotype control: n=9, or AAV9.UbC.Trastuzumab: n=12). Figure 33C shows representative bioluminescence IVIS images (week 4, left panel), total photon flux signal quantification (right panel), and Figure 33D shows Kaplan-Meier survival curves of mice bearing orthotopic brain tumors derived from MDA-MB-453 cell line after ICV treatment (AAV9.Isotype control: n=8, or AAV9.UbC.Trastuzumab: n=7). Figure 33E shows long-term trastuzumab expression quantified in perfused brain tissue by ELISA in a subset of mice shown in Figures 33C and 33D (n=5) at day 104 following ICV administration of AAV9.UbC.Trastuzumab. ***p<0.0001

[0294] Successful expression of therapeutic antibody protein in the CSF of NHPs treated with AAV9.UbC.Trastuzumab NHPs are more comparable in size to humans and have greater immunological similarity, making them the gold standard model species for evaluating AAV safety and CNS vector distribution [Gopinath, C., et al., Contemporary Animal Models For Human Gene Therapy Applications. Curr Gene Ther, 2015. 15(6): p. 531-40]. To achieve widespread vector distribution within the CNS, AAV9.UbC.Trastuzumab was administered by ICM injection (3 × 10 13AAV9 was delivered via GC / animal, n=2 / group) into the CSF of adult female Indian rhesus macaques with pre-existing AAV9 neutralizing antibodies [Hinderer, C., et al., Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna. Mol Ther Methods Clin Dev, 2014.1:p.14051]. Body weight and comprehensive serum chemistry remained within normal parameters compared to baseline measurements. No clinical signs of physical deficits or toxicity were observed throughout the study, and animals were euthanized at the scheduled endpoint on days 36-37. No gross lesions or histopathological abnormalities were observed after necropsy.

[0295] We next aimed to assess whether ICM injection of AAV9.UbC.Trastuzumab vectors resulted in detectable transgene expression in serum and CSF of treated animals as a surrogate marker of CNS gene transfer over time. Samples were collected at baseline and at weekly intervals until scheduled euthanasia (Figure 34A). Serum and CSF samples collected from NHPs previously ICM-treated with AAV9.GFP were used as negative controls. Quantification of trastuzumab levels demonstrated evidence of drug accumulation in CSF (Figure 34B), but not serum (Figure 34C), in 2 / 2 NHPs that received AAV9.UbC.Trastuzumab via ICM injection compared to control NHPs. The development of anti-drug antibodies (ADA) against trastuzumab was detected at later time points in the CSF (Figure 34D) and serum (Figure 34E) of 1 / 2 NHPs treated with AAV9.UbC.trastuzumab and may have been involved in CSF drug clearance (Figures 34D and 34E). Overall, these results confirm the successful expression of trastuzumab in the CSF of NHPs following ICM administration of AA9.UbC.trastuzumab.

[0296] Figures 34A-34E show detection of trastuzumab in the CSF of female NHPs after ICM administration of an AAV9 vector encoding trastuzumab. 13 Schematic design of experimental ICM delivery of AAV9.UbC.Trastuzumab (n=2) vectors in GC / animals is shown, informing in-life blood and CSF collection schedules and planned study termination time points. NHP No. 3 received 3×10 13 GC AAV9 vector was injected in the ICM and used as a negative control. Detection of the humanized IgG1 antibody trastuzumab in the CSF (Figure 34B) and serum (Figure 34C) of treated animals was determined by ELISA after quantification of anti-drug antibodies against human IgG and trastuzumab in the CSF (Figure 34D) and serum (Figure 34E).

[0297] ICM delivery of an AAV vector encoding trastuzumab resulted in widespread transduction and transgene expression in CNS tissues.

[0298] DNA biodistribution analysis (Figure 35A) showed that ICM AAV9.UbC.Trastuzumab (3×10 13 The results showed 100-10,000-fold higher vector GC in the brain and spinal cord compared to the heart and liver of NHPs receiving AAV9.UbC.Trastuzumab (3 × 10 GC / animal). The presence of transgene transcripts following transduction was assessed in the CNS, heart, and liver. Consistent with the DNA biodistribution data, transgene mRNA expression was 100-1,000-fold higher in the brain and spinal cord regions compared to the heart and liver of these animals (Figure 35B). 13The presence of trastuzumab transcripts in the CNS of ICM-treated NHPs (100 / animal) was confirmed via in situ hybridization using probes specific for the engineered trastuzumab version in paraffin-embedded sections from the cerebellum and occipital cortex (data not shown). Single-nucleus RNA sequencing further confirmed the presence of transgene transcripts in the cerebellum and occipital cortex from NHPs that received ICM AAV9.UbC.trastuzumab, although differential vector transduction levels were observed in these regions (0.6-1% of total trastuzumab-positive cells in the cerebellum vs. a combined 0.02-0.08% of total trastuzumab-positive cells in the occipital cortex, Figures 36A-36D). These data suggest that extracranial transduction by AAV leaking from the CSF was largely prevented in these animals because they had pre-existing levels of anti-AAV9 neutralizing antibodies prior to ICM treatment.

[0299] 35A, 35B, and 36A to 36D are 3×10 13 Gene delivery and transgene transcription throughout the CNS following ICM treatment of GC / animals with AAV9.UbC.Trastuzumab are shown. Figure 35A shows absolute quantification of AAV vector GC in NHPs 36-37 days after ICM administration of AAV9.UbC.Trastuzumab (n=2). Figure 35B shows transcripts by qPCR in selected tissues in NHPs 36-37 days after ICM administration of AAV9.UbC.Trastuzumab (n=2).

[0300] Figures 36A-36D show single nucleus RNA sequencing results demonstrating the presence of trastuzumab mRNA transcripts in the cerebellum and occipital cortex from animals ICM injected with the AAV9.UbC.Trastuzumab vector.

[0301] Next, LC-MS targeted and non-targeted proteomic approaches were used to analyze the identity of AAV-encoded trastuzumab in NHP CNS tissues (based on detection of unique trastuzumab-derived peptides and peptide mapping analysis, respectively). To reduce matrix effects and improve assay sensitivity, NHP brain homogenates were prepared under non-denaturing conditions and subjected to trastuzumab pull-down using HER2-coated magnetic beads. Ten nanograms of purified research grade trastuzumab or rituximab (anti-human CD20) antibodies were added to brain homogenates consisting of 1 mL solution containing 1 mg total protein from untreated NHP controls to provide positive or negative input controls for the pull-down assay, respectively. After incubation and recovery of the HER2-coated magnetic beads, the bead-bound antibodies were released by proteolysis and the peptide fragments were analyzed by LC-MS. For the targeted proteomics approach, the amount of trastuzumab recovered from the positive input control was used for normalization, and as expected, essentially no rituximab was recovered in the pulldown, and antibody binding to HER2 was specific. The presence of trastuzumab in brain homogenates from NHPs receiving ICM AAV9.UbC.trastuzumab was confirmed by detection of unique trastuzumab-derived peptides and peptide mapping of trastuzumab heavy and light chains (Figure 37A).

[0302] ELISA was used to further determine the abundance of human IgG protein (as a surrogate for trastuzumab) in multiple brain regions and spinal cord from NHPs receiving ICM AAV9.UbC.Trastuzumab. CNS tissue samples taken from NHPs previously ICM-treated with AAV9.GFP were used as negative controls. In the brain parenchyma (Figure 37C), levels of human IgG appeared to be consistently high (2-4 ng of trastuzumab / mg total protein) among both AAV9.UbC.Trastuzumab-treated NHPs in the cerebellum and medulla. The cerebellum is a hotsp...

Claims

1. A recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid containing a vector genome, the vector genome comprising: (a) the AAV-5′ inverted terminal repeat (ITR); (b) an expression cassette comprising a coding sequence for an anti-Her2 antibody having a heavy chain and a light chain, the expression cassette comprising: (i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody heavy chain; (ii) a nucleic acid sequence comprising SEQ ID NO:3, or a sequence at least 95% identical to SEQ ID NO:3, encoding an anti-Her2 antibody heavy chain; (iii) a furin cleavage site, (iv) a T2A element linker; (v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody light chain; (vi) a nucleic acid sequence comprising SEQ ID NO:5, or a sequence at least 95% identical to SEQ ID NO:5, encoding an anti-Her2 antibody light chain; and an expression cassette comprising a regulatory control sequence operably linked to the sequence in the expression cassette, the regulatory sequence comprising a Ubiquitin C (UbC) promoter, an optional enhancer, an intron, and a polyadenylation (polyA) sequence; (c) an AAV-3′ ITR; and

2. The rAAV of claim 1, wherein the nucleic acid sequence encoding the leader peptide of (i) comprises SEQ ID NO:

7.

3. 3. The rAAV of claim 1 or 2, wherein the nucleic acid sequence encoding the leader peptide of (v) comprises SEQ ID NO:

9.

4. The rAAV of any one of claims 1 to 3, wherein the sequence encoding the anti-Her2 antibody comprises the nucleic acid sequence of SEQ ID NO:29, or a sequence at least 95% identical to SEQ ID NO:

29.

5. The rAAV of any one of claims 1 to 4, wherein the UbC promoter has the nucleic acid sequence of SEQ ID NO:

24.

6. The rAAV of any one of claims 1 to 5, wherein the polyA sequence is an SV40 polyA sequence having the nucleic acid sequence of SEQ ID NO:

23.

7. The rAAV of any one of claims 1 to 6, wherein the intron is a chimeric intron having the nucleic acid sequence of SEQ ID NO:

22.

8. The rAAV of any one of claims 1 to 7, wherein the AAV ITRs are from AAV2.

9. The rAAV of any one of claims 1 to 8, wherein the expression cassette comprises a nucleic acid sequence of SEQ ID NO:26, or a nucleic acid sequence that is at least 99% identical to SEQ ID NO:

26.

10. 10. The rAAV of any one of claims 1 to 9, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO:25, or a sequence that is at least 99% identical to SEQ ID NO:

25.

11. A recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid containing a vector genome, the vector genome comprising: (a) the AAV-5′ inverted terminal repeat (ITR); (b) an expression cassette comprising a coding sequence for an anti-Her2 antibody having a heavy chain and a light chain, the expression cassette comprising: (i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody heavy chain; (ii) a nucleic acid sequence comprising SEQ ID NO:3, or a sequence at least 95% identical to SEQ ID NO:3, encoding an anti-Her2 antibody heavy chain; (iii) a furin cleavage site, (iv) a T2A element linker; (v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-Her2 antibody light chain; (vi) a nucleic acid sequence comprising SEQ ID NO:5, or a sequence at least 95% identical to SEQ ID NO:5, encoding an anti-Her2 antibody light chain; and an expression cassette comprising a regulatory control sequence operably linked to said sequence in said expression cassette, said regulatory sequence comprising one or more of a promoter that is a chicken beta actin promoter or a CB7 hybrid promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta actin promoter, and a chimeric intron comprising a chicken beta actin intron, and a polyA sequence that is an SV40 polyA sequence; (c) an AAV-3′ ITR; and

12. The rAAV of claim 11, wherein the promoter is a CB7 hybrid promoter having the nucleic acid sequence of SEQ ID NO:

21.

13. 13. The rAAV of claim 11 or 12, wherein the expression cassette comprises a nucleic acid sequence of SEQ ID NO:28, a nucleic acid sequence at least 99% identical to SEQ ID NO:28, a nucleic acid sequence of SEQ ID NO:2, or a nucleic acid sequence at least 99% identical to SEQ ID NO:

2.

14. 14. The rAAV of any one of claims 11 to 13, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO:27, a sequence at least 99% identical to SEQ ID NO:27, SEQ ID NO:50, a sequence at least 99% identical to SEQ ID NO:50, SEQ ID NO:1, or a sequence at least 99% identical to SEQ ID NO:

1.

15. The rAAV of any one of claims 1 to 14, wherein the AAV capsid is a clade F AAV capsid.

16. The rAAV of any one of claims 1 to 15, wherein the AAV capsid is an AAVhu68 capsid.

17. The rAAV of any one of claims 1 to 14, wherein the AAV capsid is an AAVhu95 capsid, an AAVhu96 capsid, an AAV9 capsid, or an AAVrh91 capsid.

18. 18. The rAAV of any one of claims 1 to 17, for use in treating one or more of HER2-positive cancer, HER2-positive metastatic breast cancer in the brain, or HER2-positive trastuzumab-resistant cancer.

19. The rAAV of any one of claims 1 to 18, wherein the rAAV is formulated for systemic, central nervous system, and / or intratumoral delivery.

20. A composition comprising a stock of rAAV according to any one of claims 1 to 19 and an aqueous suspension medium.

21. 21. The composition of claim 20, wherein the suspension is formulated for intrathecal delivery, optionally wherein the intrathecal delivery is intracerebroventricular (ICV) injection or intracisternomagna (ICM) injection.

22. A pharmaceutical composition comprising the rAAV of any one of claims 1 to 19 and an aqueous formulation buffer.

23. 23. The pharmaceutical composition of claim 22, formulated for intrathecal delivery.

24. 23. The pharmaceutical composition of claim 22, formulated for intracerebroventricular (ICV) or intracisternal (ICM) injection.

25. 1. A recombinant nucleic acid molecule comprising: (a) the AAV-5′ inverted terminal repeat (ITR); (b) an expression cassette comprising at least one open reading frame (ORF) comprising an anti-Her2 antibody heavy chain and an anti-Her2 antibody light chain, and a nucleic acid sequence operably linked thereto that regulates expression of the anti-Her2 heavy chain and the anti-Her2 light chain; (c) an AAV-3′ ITR; The expression cassette comprises: (i) a promoter, (A) the ubiquitin C (UbC) promoter, or (B) a promoter that is a CB7 hybrid promoter comprising a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron comprising a chicken beta actin splicing donor comprising a chicken beta actin intron and a rabbit beta globin splicing acceptor; and / or (ii) an intron that is a chimeric intron, and (iii) at least one ORF comprising a nucleic acid sequence encoding a leader peptide operably linked to an anti-Her2 heavy chain; (iv) a nucleic acid sequence comprising SEQ ID NO:3 encoding an anti-Her2 heavy chain; (v) a furin cleavage site, (vi) a T2A linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to an anti-Her2 light chain; (viii) a nucleic acid sequence comprising SEQ ID NO:5 encoding an anti-Her2 light chain; (ix) an SV40 polyadenylation (poly A) sequence, The recombinant nucleic acid molecule, wherein the expression cassette further comprises a spacer sequence.

26. 26. The recombinant nucleic acid molecule of claim 25, wherein the expression cassette comprises the nucleic acid sequence of SEQ ID NO:

26.

27. 26. The recombinant nucleic acid molecule of claim 25, comprising the nucleic acid sequence of SEQ ID NO:

25.

28. A packaging host cell comprising a recombinant nucleic acid molecule according to any one of claims 25 to 27.

29. 29. The packaging host cell of claim 28, further comprising an AAV rep coding sequence operably linked to a sequence that expresses rep proteins in the packaging host cell, an AAV capsid coding sequence operably linked to a sequence that expresses AAV capsid proteins in the packaging host cell, and helper virus functions necessary to enable packaging of the expression cassette and AAV ITRs into the AAV capsid.

30. 30. The packaging host cell of claim 28 or 29, wherein the AAV capsid is AAVhu68.

31. 30. The packaging host cell of claim 28 or 29, wherein the AAV capsid is AAV9, AAVrh91, AAVhu95, or AAVhu96.

32. 32. An rAAV production system useful for producing an rAAV according to any one of claims 1 to 19, said production system comprising a cell culture comprising a packaging host cell according to any one of claims 28 to 31.

33. 33. The rAAV production system of claim 32, wherein the AAV capsid is AAVhu68.

34. 33. The rAAV production system of claim 32, wherein the AAV capsid is AAV9, AAVrh91, AAVhu95, or AAVhu96.

35. 35. The rAAV production system of any one of claims 32 to 34, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO:

25.

36. 35. The rAAV production system of any one of claims 32 to 34, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO:27 or SEQ ID NO:

1.

37. 20. A method for treating metastatic HER2-positive cancer in the brain, the method comprising administering to a subject a suspension of the rAAV of any one of claims 1-19 in a formulation buffer.

38. 20. A method for treating metastatic breast cancer in the brain, the method comprising administering to a subject a suspension of the rAAV of any one of claims 1-19 in a formulation buffer.

39. 39. The method of claim 37 or 38, wherein the suspension is administered intrathecally.

40. 40. The method of any one of claims 37 to 39, wherein the suspension is administered in the absence of chemical or physical disruptors of the blood-brain barrier.

41. 39. The method of claim 37 or 38, wherein the suspension is administered via direct injection into a tumor bed or via an Ommaya device.

42. 23. The recombinant AAV (rAAV) of any one of claims 1 to 19 or the composition of any one of claims 20 to 22 for use in the preparation of a medicament for the treatment of metastatic HER2-positive cancer in the brain, optionally wherein said metastatic HER2-positive cancer in the brain is metastatic breast cancer in the brain.

43. 23. An anti-neoplastic regimen comprising administering an rAAV according to any one of claims 1 to 19, a composition according to any one of claims 20 to 22, or a pharmaceutical composition according to claim 23 or 24 in combination with a biological drug, a small molecule, an anti-neoplastic agent, radiation, and / or a chemotherapeutic agent.

44. 11. A method for treating a HER2-positive cancer that is resistant to trastuzumab, the method comprising administering to a subject a suspension of the rAAV of claim 1 in a formulation buffer.

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