Systemic AAV9-IFNβ gene delivery treats highly invasive glioblastoma.

Neuro-Oncology Neuro-Oncology Neuro-Oncology 18(11), 1508–1518, 2016

doi:10.1093/neuonc/now097 Neuro-Oncology 2016; 0, 1 – 11, doi:10.1093/neuonc/now097 Advance Access date 18 May 2016

Systemic AAV9-IFNb gene delivery treats highly invasive glioblastoma Dwijit GuhaSarkar, Qin Su, Guangping Gao, and Miguel Sena-Esteves Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts (D.G., Q.S., G.G., M.S.-E.); Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts (D.G., M.S.-E.); Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts (G.G.) Corresponding Author: Miguel Sena-Esteves, 368 Plantation Street, AS6-2055, Worcester, MA 01605 ([email protected]).

Background. Complete surgical removal of all glioblastoma (GBM) cells is impossible due to extensive infiltration into brain parenchyma that ultimately leads to tumor recurrence. The current standard of care affords modest improvements in survival. New therapeutic interventions are needed to prevent recurrence. Local AAV-hIFNb gene delivery to the brain was previously shown to eradicate noninvasive orthotopic U87 tumors in mice. However, widespread CNS gene delivery may be necessary to treat invasive GBMs. Here we investigated the therapeutic effectiveness of systemically infused AAV9-hIFNb against an invasive orthotopic GBM8 model. Methods. Mice bearing human GBM8 brain tumors expressing firefly luciferase (Fluc) were treated systemically with different doses of scAAV9-hIFNb vector. Therapeutic efficacy was assessed by sequential bioluminescence imaging of tumor Fluc activity and animal survival. Brains were analyzed post mortem for the presence and appearance of tumors. Two transcriptionally restricted AAV vectors were used to assess the therapeutic contribution of peripheral hIFNb. Results. Systemic infusion of scAAV9-hIFNb vector induced complete regression of established GBM8 tumors in a dose-dependent manner. The efficacy of this approach was also dependent on the stage of tumor growth at the time of treatment. We also showed that peripherally produced hIFNb contributed considerably to the therapeutic effect of scAAV9-hIFNb. A comparative study of systemic and unilateral intracranial delivery of scAAV9-hIFNb in a bilateral GBM8 tumor model showed the systemic route to be the most effective approach for treating widely dispersed tumors. Conclusions. Systemic delivery of AAV9-IFNb is an attractive approach for invasive and multifocal GBM treatment. Keywords: adeno associated virus (AAV), glioblastoma (GBM), interferon-beta (IFNb), mouse orthotopic xenograft, systemic AAV gene therapy.

Glioblastoma (GBM) is the most common1 and aggressive (WHO grade IV) primary brain tumor.2 These tumors are characterized by aggressive invasiveness, long-distance migration, and neovascularization. The standard-of-care treatment involves surgical resection, followed by radiation and chemotherapy with temozolomide. Complete surgical resection of tumor is almost impossible because of macroscopically indistinguishable, diffuse GBM cell infiltrates far from the main tumor mass. This results in tumor recurrence, which is often radio- or chemotherapy resistant, eventually causing death. Overall median survival of GBM patients post diagnosis is 14.6 months.3 Hence, new therapeutic approaches are needed to prevent tumor recurrence. Interferon beta (IFNb), a cytokine secreted from cells upon viral infection, activates several genes downstream through Janus kinases (JAKs) and signal transducers and activators of

transcription (STATs).4 Although IFNb is best known for its antiviral immune modulatory roles, it also has antitumor properties. In addition to direct cytostatic and proapoptotic effects on cancer cells, 5,6 IFNb also has antiangiogenic 7,8 and immune-stimulatory9,10 properties. In addition, IFNb has been shown to potentiate the effect of some chemotherapeutic drugs.11 Previously, a phase 1 clinical trial on newly diagnosed GBM patients showed improved survival in patients treated with IFNb as an adjuvant to the standard of care treatment with minimal adverse effects.12 However, given the relatively short half-life of IFNb,13 achieving greater therapeutic benefit is challenging. Gene therapy approaches may bypass this limitation through continuous expression of INFb in transduced cells. Moreover, as IFNb is a secreted protein, transduction of a small number of endogenous cells may be sufficient to achieve

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GuhaSarkar et al.: Systemic AAV9-IFNb treatment for glioma GuhaSarkar et al.: Systemic AAV9-IFNb treatment for glioma

a widespread BkFQ. Mouse therapeutic HPRT1 expression effect. Previously, was used we as have an internal shown reference that local expression gene toof normalize human IFNball (hIFNb) values from(Assay a recombiID: Mm00446968_m1; nant adeno-associated Applied viralBiosystems). (AAV) vectorMean successfully expression inhibits values growth for of PBSintracranial group animals human were U87 considered tumors through to be background gene delivU87 brain tumors grow rapidly noise ery to and normal thuscells were in the subtracted brain.14 from all values. in the mouse brain, but mostly as large spheroids with distinct borders, and thus do not reproduce the invasiveness and Quantification of Human IFNbare in responsible Cell-conditioned Media migratory properties of GBM that for recurrence. and Mouse Plasma To test the effectiveness of AAV-hIFNb gene therapy in a more realistic we carried outmedium studies and usingmouse GBM8 plasma cells, which hIFNb inmodel, conditioned growth was upon transplantation mouse brainand produce diffuse tumors measured using ELISAinto assays (41 410 41 415, respectively; 15 with extensive infiltration and long-distance migration. PBL Assay Science). Conditioned growth media were collected potential approach to counteractMouse the properties of GBM for AELISA 48 hours after transfection. plasma was colcells that limits the efficacy of local Media interventions is to generate lected 1 month after AAV infusion. and plasma were dia disperse of endogenous cells expressing IFNb preluted 1:10network and 1:4,000 in PBS, respectively, for to ELISA vent single tumor cell infiltration and growth (ie, matching the measurements. delivery modality to the disease characteristics). Intravascular infusion of AAV9 vectors achieves widespread gene delivery to 16 Histological Analysis and large animals.17,18 Therefore, systemic the CNS of mice delivery of an AAV9-hIFNb may be sections effective(5 formm) generatBrain cryosections (20 mm)vector and paraffin were ing a for widely distributed CNS network to combat the used invasive used histological studies. The primary antibodies for and migratory properties were of GBM. Here we tested thisanti-GFP concept immunohistochemistry rabbit monoclonal in an orthotopic model of invasive human anti-Olig2 GBM. (1:1000, G10362,xenograft Invitrogen) and rabbit monoclonal (1:250, ab109186, Abcam). Biotinylated goat anti-rabbit IgG (1:1000, BA-1000, Vector Laboratories) was used as secondary Materials and Methods antibody. VECTASTAIN Elite ABC Kit (PK-6100, Vector Laboratories) and DAB substrate kit (SK-4100, Vector Laboratories) were Cell Culture used for immunohistochemical detection and Mayer′ s hemaGBM8 were as a kind gift from Dr.Cryosections Samuel Rabkin toxylincells (Sigma) counterstain. (20(Massachumm) were setts General Hospital, Boston, Massachusetts). These cells used to detect human GBM8-Fluc cells in the brain by immunowere transduced with the lentivirus vector CSCW2-Flucfluorescence using mouse anti-human nuclei (1:150, 14 todetection generate with GBM8-Fluc cells conIRES-mCherry asMillipore) describedand MAB4383, EMD Alexa-fluor 594 stitutively expressing firefly luciferase. Cells were grown as goat anti-mouse (1: 2000, A-11020, Invitrogen). Sections neurospheres in neurobasal ′ media (21103-049, Gibco) supplewere also stained with 4 ,6-diamidino-2-phenylindole (DAPI) mented with 3 mMFisher L-glutamine (25-005-CI, Mediatech), 1x (PI-62247, Thermo Scientific). B27 supplement (17504-044, Gibco), 0.5x N2 supplement (17502-048, Gibco), 2 mg/mL heparin (H3400, Sigma-Aldrich), 1x antibiotic-antimycotic solution (30-004-CI, Mediatech) and Graphs and Statistical Analysis 1x amphotericin B (30-003-CF, Mediatech), ng/mL recombiKaplan-Meier survival curves were plotted in20 Prism 6 (GraphPad nant human bFGF (100-18B, PeproTech), and 20 ng/mL of Software), and the Mantel-Cox log-rank test was used for statrecombinant human EGF (AF-100-15, istical analysis. Bar graphs were also PeproTech). plotted using the same software. And an unpaired 2-tailed t test was used for statistical analysis. Calculated P values were defined as the probability Animals of null hypothesis being true; *P , .05; **P , .01; ***P , .001; Sixmale athymic ****Pto ,eight .0001;week-old ns, not significant (P .nude .05). mice were obtained from the National Cancer Institute for this study. All animal studies were approved by the University of Massachusetts MedResults ical School Institutional Animal Care and Use Committee following guidelines set forth by the NIH’s Guide for the Care Dose-dependent Therapeutic Response With Systemic and Use of Laboratory Animals (https://grants.nih.gov/grants/ Delivery of scAAV9/CB-hIFNb olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf).19 To assess the therapeutic effectiveness of systemically delivered scAAV9/CB-hIFNb, athymic nude mice were treated with Orthotopic Xenografting different vector doses (1 × 1011, 3 × 1011, and 1 × 1012 gc) or Two prior to implantation into the mice, the medium of PBS 2days weeks after GBM8-Fluc tumor implantation. We meaGBM8-Fluc was replaced with fresh medium. Onsome the day sured TABS cells by live bioluminescence imaging to gain inof injection, GBM8-Fluc cells were dissociated single cell sight into changes in tumor growth kineticsinto in athe mouse ′ suspension pipetting. Cells were washed twicecontrol in Dulbecco brain after by AAV treatment. In PBS-injected mice, s TABS increased exponentially until the animals reached huphosphate-buffered saline (PBS; 14190-250, Gibco) andthe resusmane endpoint defined by .15% loss in 000 maximum pended in the same to a concentration of 50 cells/mL.body One

weight. In suspension the 2 top doses scAAV9/CB-hIFNb, TABS decreased mL of cell wasof injected stereotaxically into the left over time The and was indistinguishable from (week-1 sigstriatum. stereotaxic coordinates forbaseline tumor implantation nal) by 2 weekswere after(in treatment weeksML: after from bregma mm): AP:(4+0.5, 2.0tumor (left) implanand DV tation). Thissurface: remained unchanged over time (Fig. 1A and B). from brain 22.5. Bilateral tumors were generated by response. TABS deTreatment × 1011 gc showed injecting 50with 000 1GBM8-Fluc cells intopartial both striata. creased to baseline levels in 2 mice while increasing over time in 3 mice, albeit with Production, different kinetics in the PBS control AAV Vector Design, and than Delivery group (Fig. 1B). All The recombinant AAV9sdose used in thewas study were selfscAAV9/CB-hIFNb response also reflected in complementary (sc) vectors. scAAV9/CB-hIFNb and long-term survival with 100%, 88.89%, and 20% of scAAV9/ animals CB-hIFNb -miRBS-1-122 vectorsimplantation encode human alive at 244 days post tumor in 1interferon-b × 1012, 3 × 11 the chicken 11 b-actin promoter and cytomegalovirus enunder 10 , and 1 ×10 gc dose groups, respectively (Fig. 1C). All anhancer (CB promoter) and carry a rabbit beta-globin polyadenyimals from the PBS control group reached the humane endlation (RBGpA) 42 signal. scAAV9/CB-hIFNb-miRBS-1-122 point between and The 49 days with a median survival of vector 3 copies of miR-1 and miR-122 binding sites 46 dayscarries after tumor implantation. 20 ′ (miRBS) in the 3 untranslated region described. The Histological analysis of the brains from as animals that survived scAAV9/TBG-hIFNb vector carries a thyroxin-binding globulin to 244 days revealed normal histology with no microscopic ev21 (TBG) promoter drive gene The idence of tumorsto (Fig. 1D, liver-specific Supplementary Fig.expression. S1). Interestingly, scAAV9/CB-EGFP and scAAV9/TBG-EGFP vectors encode in animals that succumbed to disease progression in the en1× 11 hanced green fluorescence protein (EGFP). 10 gc group, there were large tumors with sharply defined AAV9 to vectors were produced at thecomposed University of borders normal tissue that were of Massachunumerous setts Medical School Gene Therapy Center Viral Vector Core as well-defined tumorlets (arrows in Fig. 1D). This is in contrast 22 Vector titers were determined by quantitative PCR described. to the diffuse nature of GBM8 tumors in mouse brain found in (qPCR) vectorgroup genomes using the following primers and the PBSof control (Fig. 1D). probe specific for RBGpA (Eurofins): Primer1: 5′ -GCCAAAAATTATGGGGACAT-3′ ;

′ Therapeutic Is Dependent on Tumor Growth -ATTCCAACACACTATTGCAATG-3 ; Primer2: 5′Outcome Phase at the Time of Treatment Probe: 6FAM-ATGAAGCCCCTTGAGCATCTGACTTCT-TAMRA

To assess the effect of tumor growth the therapeutic For systemic administration, AAV9 phase vectorsonwere injected via 11 gc (minimum effective dose) of scAAV9/ efficacy 3 ×10 the tail vein in a total volume of 200 mL in PBS. In the intracraCB-hIFNb was infused at 2,7.6 3,×3.5 hourscopies after week-3 genome (gc) of nial treatment paradigm, 109(�84 treatment), and 4 weeks implantation. scAAV9/CB-hIFNb vector after wereGBM8-Fluc infused in tumor 2 mL at 200 nL/min Treatment week 2 and week 3 prevented as in the sameatstereotaxic coordinates used for tumor tumor growth implantasuggested by TABS assessment over time (Fig. 2A). More imtion. For control groups, an equal volume of PBS was injected portantly, thisforultimately resulted in long-term survival of into the mice all of the experiments. 100% and 88.9% of mice to 244 days after tumor implantation (Fig. 2B). Treatment at 3.5 and 4 weeks had a modest imLive Bioluminescence pact on TABS increase Imaging over time (Fig. 2A). Nonetheless, it Imaging in of significant tumor-associated signal (TABS) resulted increasesbioluminescence in median survival to 60 and wasdays, performed using compared the Xenogen IVIS 61 respectively, with 46 100 daysimaging for PBS system control (PerkinElmer) 3 minutes after intraperitoneal administration mice. The maximum survivals were increased to 150 and of D-luciferin (4.5 mg). Image analysis using 80 days, respectively, compared with 54was daysperformed for PBS controls Living2B). Image software (PerkinElmer). (Fig. Analysis of tumor burden by TABS at the treatment time points revealed tumors unchanged bePreparation of that Tissue DNA remained and RNA largely and Quantification tween weeks 2 and 3 but increased thereafter of Vector Genomes and hIFNb Transcripts (Fig. 2C and D). Immunostaining of tumor sections for human nuclear antigen Total DNA was extracted using the DNeasy Blood & Tissue and GBM-specific OLIG2 marker at the time of treatment corkit (QIAGEN). DNA was diluted to a final concentration of roborated this observation (Fig. 2E, Supplementary Fig S2). 50– 100 ng/mLfor vector genome quantification by qPCR using These results suggest a correlation between therapeutic efficaRBGpolyA specific primers and probe. cy of scAAV9/CB-hIFNb and tumor growth phase. Tissue RNA was isolated using TRIzol (15596-018, Invitrogen) and Direct-zol RNA MiniPrep (R2052, Zymo Research Corporation). was treated with TURBO DNase (AM1907, Systemically RNA Delivered AAV9 Primarily Transduces Ambion) for 30 minutes at 378C prior to reverse transcription Astrocytes and Endothelial Cells in the using High Capacity RNA-to-cDNA kit (4387406, Applied BiosysGlioblastoma-bearing Brain tems). Quantitative PCRMouse was performed with the following Tumor-bearing primers and probe mice for were hIFNb injected (IDT): systemically Primer-1:with 5 ′ -GCAATTGAA 3 × 1011 gc ′ ′ ; Primer-2: -TCATAGATGGTCAATGCGGC-3 TGGGAGGCTTG-3 of scAAV9/CB-EGFP vector to 5 determine the cell types trans-′ ; ′ Probe: 5 -/6-FAM/TGTCAAAGT/ZEN/TCATCCTGTCCTTGAGGC/3IA duced and thus were likely mediators of the therapeutic effect.

Neuro-Oncology1509 Neuro-Oncology 2 of 11 Neuro-Oncology 3 of 11


BkFQ. a widespread Mouse therapeutic HPRT1 expression effect. Previously, was used we as have an internal shown that local expression reference gene toof normalize human IFNball (hIFNb) values from(Assay a recombiID: nant adeno-associated Mm00446968_m1; Applied viralBiosystems). (AAV) vectorMean successfully expression inhibits valgrowth ues for of PBSintracranial group animals human were U87 considered tumors through to be background gene delivU87 brain tumors grow rapidly ery to and noise normal thuscells were in the subtracted brain.14 from all values. in the mouse brain, but mostly as large spheroids with distinct borders, and thus do not reproduce the invasiveness and Quantification of Human IFNbare in responsible Cell-conditioned Media migratory properties of GBM that for recurrence. and Mouse Plasma To test the effectiveness of AAV-hIFNb gene therapy in a more realistic we carried outmedium studies and usingmouse GBM8 plasma cells, which hIFNb inmodel, conditioned growth was upon transplantation mouse brainand produce diffuse tumors measured using ELISAinto assays (41 410 41 415, respectively; 15 with extensive infiltration and long-distance migration. PBL Assay Science). Conditioned growth media were collected potential approach to counteractMouse the properties of GBM for AELISA 48 hours after transfection. plasma was colcells that limits the efficacy of local Media interventions is to generate lected 1 month after AAV infusion. and plasma were dia disperse of endogenous cells expressing IFNb preluted 1:10network and 1:4,000 in PBS, respectively, for to ELISA vent single tumor cell infiltration and growth (ie, matching the measurements. delivery modality to the disease characteristics). Intravascular infusion of AAV9 vectors achieves widespread gene delivery to 16 Histological Analysis and large animals.17,18 Therefore, systemic the CNS of mice delivery of an AAV9-hIFNb may be sections effective(5 formm) generatBrain cryosections (20 mm)vector and paraffin were ing a for widely distributed CNS network to combat the used invasive used histological studies. The primary antibodies for and migratory properties were of GBM. Here we tested thisanti-GFP concept immunohistochemistry rabbit monoclonal in an orthotopic model of invasive human anti-Olig2 GBM. (1:1000, G10362,xenograft Invitrogen) and rabbit monoclonal (1:250, ab109186, Abcam). Biotinylated goat anti-rabbit IgG (1:1000, BA-1000, Vector Laboratories) was used as secondary Materials and Methods antibody. VECTASTAIN Elite ABC Kit (PK-6100, Vector Laboratories) and DAB substrate kit (SK-4100, Vector Laboratories) were Cell Culture used for immunohistochemical detection and Mayer′ s hemaGBM8 were as a kind gift from Dr.Cryosections Samuel Rabkin toxylincells (Sigma) counterstain. (20(Massachumm) were setts General Hospital, Boston, Massachusetts). These cells used to detect human GBM8-Fluc cells in the brain by immunowere transduced with the lentivirus vector CSCW2-Flucfluorescence using mouse anti-human nuclei (1:150, 14 todetection generate with GBM8-Fluc cells conIRES-mCherry asMillipore) describedand MAB4383, EMD Alexa-fluor 594 stitutively expressing firefly luciferase. Cells were grown as goat anti-mouse (1: 2000, A-11020, Invitrogen). Sections neurospheres in neurobasal ′ media (21103-049, Gibco) supplewere also stained with 4 ,6-diamidino-2-phenylindole (DAPI) mented with 3 mMFisher L-glutamine (25-005-CI, Mediatech), 1x (PI-62247, Thermo Scientific). B27 supplement (17504-044, Gibco), 0.5x N2 supplement (17502-048, Gibco), 2 mg/mL heparin (H3400, Sigma-Aldrich), 1x antibiotic-antimycotic solution (30-004-CI, Mediatech) and Graphs and Statistical Analysis 1x amphotericin B (30-003-CF, Mediatech), ng/mL recombiKaplan-Meier survival curves were plotted in20 Prism 6 (GraphPad nant human bFGF (100-18B, PeproTech), and 20 ng/mL of Software), and the Mantel-Cox log-rank test was used for statrecombinant human EGF (AF-100-15, istical analysis. Bar graphs were also PeproTech). plotted using the same software. And an unpaired 2-tailed t test was used for statistical analysis. Calculated P values were defined as the probability Animals of null hypothesis being true; *P , .05; **P , .01; ***P , .001; Sixmale athymic ****Pto ,eight .0001;week-old ns, not significant (P .nude .05). mice were obtained from the National Cancer Institute for this study. All animal studies were approved by the University of Massachusetts MedResults ical School Institutional Animal Care and Use Committee following guidelines set forth by the NIH’s Guide for the Care Dose-dependent Therapeutic Response With Systemic and Use of Laboratory Animals (https://grants.nih.gov/grants/ Delivery of scAAV9/CB-hIFNb olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf).19 To assess the therapeutic effectiveness of systemically delivered scAAV9/CB-hIFNb, athymic nude mice were treated with Orthotopic Xenografting different vector doses (1 × 1011, 3 × 1011, and 1 × 1012 gc) or Two prior to implantation into the mice, the medium of PBS 2days weeks after GBM8-Fluc tumor implantation. We meaGBM8-Fluc was replaced with fresh medium. Onsome the day sured TABS cells by live bioluminescence imaging to gain inof injection, GBM8-Fluc cells were dissociated single cell sight into changes in tumor growth kineticsinto in athe mouse ′ suspension pipetting. Cells were washed twicecontrol in Dulbecco brain after by AAV treatment. In PBS-injected mice, s TABS increased exponentially until the animals reached huphosphate-buffered saline (PBS; 14190-250, Gibco) andthe resusmane endpoint defined by .15% loss in 000 maximum pended in the same to a concentration of 50 cells/mL.body One 1510 2 of 11 Neuro-Oncology

weight. In suspension the 2 top doses scAAV9/CB-hIFNb, TABS decreased mL of cell wasof injected stereotaxically into the left over time The and was indistinguishable from (week-1 sigstriatum. stereotaxic coordinates forbaseline tumor implantation nal) by 2 weekswere after(in treatment weeksML: after from bregma mm): AP:(4+0.5, 2.0tumor (left) implanand DV tation). Thissurface: remained unchanged over time (Fig. 1A and B). from brain 22.5. Bilateral tumors were generated by response. TABS deTreatment × 1011 gc showed injecting 50with 000 1GBM8-Fluc cells intopartial both striata. creased to baseline levels in 2 mice while increasing over time in 3 mice, albeit with Production, different kinetics in the PBS control AAV Vector Design, and than Delivery group (Fig. 1B). All The recombinant AAV9sdose used in thewas study were selfscAAV9/CB-hIFNb response also reflected in complementary (sc) vectors. scAAV9/CB-hIFNb and long-term survival with 100%, 88.89%, and 20% of scAAV9/ animals CB-hIFNb -miRBS-1-122 vectorsimplantation encode human alive at 244 days post tumor in 1interferon-b × 1012, 3 × 11 the chicken 11 b-actin promoter and cytomegalovirus enunder 10 , and 1 ×10 gc dose groups, respectively (Fig. 1C). All anhancer (CB promoter) and carry a rabbit beta-globin polyadenyimals from the PBS control group reached the humane endlation (RBGpA) 42 signal. scAAV9/CB-hIFNb-miRBS-1-122 point between and The 49 days with a median survival of vector 3 copies of miR-1 and miR-122 binding sites 46 dayscarries after tumor implantation. 20 ′ (miRBS) in the 3 untranslated region described. The Histological analysis of the brains from as animals that survived scAAV9/TBG-hIFNb vector carries a thyroxin-binding globulin to 244 days revealed normal histology with no microscopic ev21 (TBG) promoter drive gene The idence of tumorsto (Fig. 1D, liver-specific Supplementary Fig.expression. S1). Interestingly, scAAV9/CB-EGFP and scAAV9/TBG-EGFP vectors encode in animals that succumbed to disease progression in the en1× 11 hanced green fluorescence protein (EGFP). 10 gc group, there were large tumors with sharply defined AAV9 to vectors were produced at thecomposed University of borders normal tissue that were of Massachunumerous setts Medical School Gene Therapy Center Viral Vector Core as well-defined tumorlets (arrows in Fig. 1D). This is in contrast 22 Vector titers were determined by quantitative PCR described. to the diffuse nature of GBM8 tumors in mouse brain found in (qPCR) vectorgroup genomes using the following primers and the PBSof control (Fig. 1D). probe specific for RBGpA (Eurofins): Primer1: 5′ -GCCAAAAATTATGGGGACAT-3′ ;

′ Therapeutic Is Dependent on Tumor Growth -ATTCCAACACACTATTGCAATG-3 ; Primer2: 5′Outcome Phase at the Time of Treatment Probe: 6FAM-ATGAAGCCCCTTGAGCATCTGACTTCT-TAMRA

To assess the effect of tumor growth the therapeutic For systemic administration, AAV9 phase vectorsonwere injected via 11 gc (minimum effective dose) of scAAV9/ efficacy 3 ×10 the tail vein in a total volume of 200 mL in PBS. In the intracraCB-hIFNb was infused at 2,7.6 3,×3.5 hourscopies after week-3 genome (gc) of nial treatment paradigm, 109(�84 treatment), and 4 weeks implantation. scAAV9/CB-hIFNb vector after wereGBM8-Fluc infused in tumor 2 mL at 200 nL/min Treatment week 2 and week 3 prevented as in the sameatstereotaxic coordinates used for tumor tumor growth implantasuggested by TABS assessment over time (Fig. 2A). More imtion. For control groups, an equal volume of PBS was injected portantly, thisforultimately resulted in long-term survival of into the mice all of the experiments. 100% and 88.9% of mice to 244 days after tumor implantation (Fig. 2B). Treatment at 3.5 and 4 weeks had a modest imLive Bioluminescence pact on TABS increase Imaging over time (Fig. 2A). Nonetheless, it Imaging in of significant tumor-associated signal (TABS) resulted increasesbioluminescence in median survival to 60 and wasdays, performed using compared the Xenogen IVIS 61 respectively, with 46 100 daysimaging for PBS system control (PerkinElmer) 3 minutes after intraperitoneal administration mice. The maximum survivals were increased to 150 and of D-luciferin (4.5 mg). Image analysis using 80 days, respectively, compared with 54was daysperformed for PBS controls Living2B). Image software (PerkinElmer). (Fig. Analysis of tumor burden by TABS at the treatment time points revealed tumors unchanged bePreparation of that Tissue DNA remained and RNA largely and Quantification tween weeks 2 and 3 but increased thereafter of Vector Genomes and hIFNb Transcripts (Fig. 2C and D). Immunostaining of tumor sections for human nuclear antigen Total DNA was extracted using the DNeasy Blood & Tissue and GBM-specific OLIG2 marker at the time of treatment corkit (QIAGEN). DNA was diluted to a final concentration of roborated this observation (Fig. 2E, Supplementary Fig S2). 50– 100 ng/mLfor vector genome quantification by qPCR using These results suggest a correlation between therapeutic efficaRBGpolyA specific primers and probe. cy of scAAV9/CB-hIFNb and tumor growth phase. Tissue RNA was isolated using TRIzol (15596-018, Invitrogen) and Direct-zol RNA MiniPrep (R2052, Zymo Research Corporation). was treated with TURBO DNase (AM1907, Systemically RNA Delivered AAV9 Primarily Transduces Ambion) for 30 minutes at 378C prior to reverse transcription Astrocytes and Endothelial Cells in the using High Capacity RNA-to-cDNA kit (4387406, Applied BiosysGlioblastoma-bearing Brain tems). Quantitative PCRMouse was performed with the following primers and probe Tumor-bearing mice for were hIFNb injected (IDT): systemically Primer-1:with 5 ′ -GCAATTGAA 3 × 1011 gc ′ ′ ; Primer-2: -TCATAGATGGTCAATGCGGC-3 TGGGAGGCTTG-3 of scAAV9/CB-EGFP vector to 5 determine the cell types trans-′ ; ′ Probe: 5 -/6-FAM/TGTCAAAGT/ZEN/TCATCCTGTCCTTGAGGC/3IA duced and thus were likely mediators of the therapeutic effect. Neuro-Oncology 3 of 11


BkFQ. Mouse HPRT1 expression was used as an internal reference gene to normalize all values (Assay ID: Mm00446968_m1; Applied Biosystems). Mean expression values for PBS group animals were considered to be background noise and thus were subtracted from all values.

Quantification of Human IFNb in Cell-conditioned Media and Mouse Plasma hIFNb in conditioned growth medium and mouse plasma was measured using ELISA assays (41 410 and 41 415, respectively; PBL Assay Science). Conditioned growth media were collected for ELISA 48 hours after transfection. Mouse plasma was collected 1 month after AAV infusion. Media and plasma were diluted 1:10 and 1:4,000 in PBS, respectively, for ELISA measurements.

Histological Analysis Brain cryosections (20 mm) and paraffin sections (5 mm) were used for histological studies. The primary antibodies used for immunohistochemistry were rabbit monoclonal anti-GFP (1:1000, G10362, Invitrogen) and rabbit monoclonal anti-Olig2 (1:250, ab109186, Abcam). Biotinylated goat anti-rabbit IgG (1:1000, BA-1000, Vector Laboratories) was used as secondary antibody. VECTASTAIN Elite ABC Kit (PK-6100, Vector Laboratories) and DAB substrate kit (SK-4100, Vector Laboratories) were used for immunohistochemical detection and Mayer′ s hematoxylin (Sigma) as counterstain. Cryosections (20 mm) were used to detect human GBM8-Fluc cells in the brain by immunofluorescence using mouse anti-human nuclei (1:150, MAB4383, EMD Millipore) and detection with Alexa-fluor 594 goat anti-mouse (1: 2000, A-11020, Invitrogen). Sections were also stained with 4′ ,6-diamidino-2-phenylindole (DAPI) (PI-62247, Thermo Fisher Scientific).

weight. In the 2 top doses of scAAV9/CB-hIFNb, TABS decreased over time and was indistinguishable from baseline (week-1 signal) by 2 weeks after treatment (4 weeks after tumor implantation). This remained unchanged over time (Fig. 1A and B). Treatment with 1 × 1011 gc showed partial response. TABS decreased to baseline levels in 2 mice while increasing over time in 3 mice, albeit with different kinetics than in the PBS control group (Fig. 1B). The scAAV9/CB-hIFNb dose response was also reflected in long-term survival with 100%, 88.89%, and 20% of animals alive at 244 days post tumor implantation in 1 × 1012, 3 × 1011, and 1 ×1011 gc dose groups, respectively (Fig. 1C). All animals from the PBS control group reached the humane endpoint between 42 and 49 days with a median survival of 46 days after tumor implantation. Histological analysis of the brains from animals that survived to 244 days revealed normal histology with no microscopic evidence of tumors (Fig. 1D, Supplementary Fig. S1). Interestingly, in animals that succumbed to disease progression in the 1 × 1011 gc group, there were large tumors with sharply defined borders to normal tissue that were composed of numerous well-defined tumorlets (arrows in Fig. 1D). This is in contrast to the diffuse nature of GBM8 tumors in mouse brain found in the PBS control group (Fig. 1D).

Therapeutic Outcome Is Dependent on Tumor Growth Phase at the Time of Treatment

To assess the effect of tumor growth phase on the therapeutic efficacy 3 ×10 11 gc (minimum effective dose) of scAAV9/ CB-hIFNb was infused at 2, 3, 3.5 (�84 hours after week-3 treatment), and 4 weeks after GBM8-Fluc tumor implantation. Treatment at week 2 and week 3 prevented tumor growth as suggested by TABS assessment over time (Fig. 2A). More importantly, this ultimately resulted in long-term survival of 100% and 88.9% of mice to 244 days after tumor implantation (Fig. 2B). Treatment at 3.5 and 4 weeks had a modest imGraphs and Statistical Analysis pact on TABS increase over time (Fig. 2A). Nonetheless, it Kaplan-Meier survival curves were plotted in Prism 6 (GraphPad resulted in significant increases in median survival to 60 and Software), and the Mantel-Cox log-rank test was used for stat- 61 days, respectively, compared with 46 days for PBS control istical analysis. Bar graphs were also plotted using the same mice. The maximum survivals were increased to 150 and Fig. 1. Systemic delivery of scAAV9/CB-hIFNb dose-dependent response in an compared orthotopic GBM8 mouse (A) software. And an unpaired 2-tailed t testproduces was used for statisti- therapeutic 80 days, respectively, with xenograft 54 days for PBSmodel. controls Bioluminescence imaging of tumor burden in representative mice from each group for weeks 2– 6 after tumor implantation. Regions of interest cal analysis. Calculated P values were defined as the probability (Fig. 2B). (ROI) used for signal being quantification shown circles. (B).001; Tumor-associated bioluminescence signal (TABS) was assessed weekly up to of null hypothesis true; *Pare , .05; **Pas,red .01; ***P , Analysis of tumor burden by TABS at the treatment time 16 weeks and represented as fold change over signal at 1 week after tumor injection. Data are shown as mean+SD. Black arrow on the x-axis ****P , .0001; ns, not significant (P . .05). points revealed that tumors remained largely unchanged beindicates time of treatment. Treatment groups are represented as follows: 1 ×10 12 gc (red), 3 ×1011 gc (blue), 1 ×1011 gc (green), and tween weeks 2 respondent and 3 but increased 2C and 11 phosphate-buffered saline (PBS) control group (black); “s1” and “s2,” nonrespondent and subgroups, thereafter respectively.(Fig. For the 1 ×10D). Immunostaining of tumor sections for human nuclear antigen gc group, the TABS curves for complete (dark green) and partial (light green) responders are plotted separately.. (C) Kaplan-Meier survival Results 12 treatment corandSurvival GBM-specific OLIG2 marker at the time curves for different treatment groups show dose-dependent survival benefit. curves are identified as follows: 1×10of gc (red), 3×1011 this,observation Fig S2). Dose-dependent gc (blue), 1 ×1011 gcTherapeutic (green), and Response PBS control With group Systemic (black); log-rankroborated P values: ****P .0001; ***P ,(Fig. .001.2E, (D) Supplementary Representative images of These suggest a correlation between efficahematoxylin-and-eosin staining of mouse brain sections at the endpoints. Top results row depicts representative brain sectionstherapeutic from different dose Delivery of scAAV9/CB-hIFNb groups. Bottom row shows the magnified views of the corresponding areas as “a,” “b,”and “c,”tumor and “d”growth in the top row. Black arrows cy ofindicated scAAV9/CB-hIFNb phase. To assess the therapeutic effectiveness ofbrains systemically deliv- at the humane endpoints on day 44 (PBS control) and day-111 (1 × point to tumorlets with distinct borders. The were collected 11 scAAV9/CB-hIFNb, athymic nude mice were ered with12 gc). Scale bars represent 100 mm. Abbreviation: n, number of animals 10 gc), or at the study endpoint on day-244 (3 ×1011 treated gc and 1×10 different vector doses (1 × 1011, 3 × 1011, and 1 × 1012 gc) or Systemically Delivered AAV9 Primarily Transduces in the group. PBS 2 weeks after GBM8-Fluc tumor implantation. We meaAstrocytes and Endothelial Cells in the sured TABS by live bioluminescence imaging to gain some inMouse Brain dose, the therapeutic Based on morphology 3Agrowth and B), kinetics it appeared thatmouse most Glioblastoma-bearing results suggest that, at the intermediate sight into changes in (Fig. tumor in the EGFP-positive cells were astrocytes and endothelial cells Tumor-bearing effect of scAAV9/CB-hIFNb is verysystemically likely achieved brain after AAV treatment. In PBS-injected control mice, mice were injected withthrough 3 × 1011exgc found primarilyexponentially in cortex anduntil periventricular pression of hIFNbvector in normal astrocytes and vascular TABS increased the animalsregions reached(Fig. the 3B), hu- of scAAV9/CB-EGFP to determine the cell types transwith transduced the striatum or tumor. These endothelia. manefewer endpoint definedcells by in .15% loss in maximum body duced and thus were likely mediators of the therapeutic effect. Neuro-Oncology1511 Neuro-Oncology 4 of 11 Neuro-Oncology 3 of 11


a widespread therapeutic effect. Previously, we have shown that local expression of human IFNb (hIFNb) from a recombinant adeno-associated viral (AAV) vector successfully inhibits growth of intracranial human U87 tumors through gene delivery to normal cells in the brain.14 U87 brain tumors grow rapidly in the mouse brain, but mostly as large spheroids with distinct borders, and thus do not reproduce the invasiveness and migratory properties of GBM that are responsible for recurrence. To test the effectiveness of AAV-hIFNb gene therapy in a more realistic model, we carried out studies using GBM8 cells, which upon transplantation into mouse brain produce diffuse tumors with extensive infiltration and long-distance migration.15 A potential approach to counteract the properties of GBM cells that limits the efficacy of local interventions is to generate a disperse network of endogenous cells expressing IFNb to prevent single tumor cell infiltration and growth (ie, matching the delivery modality to the disease characteristics). Intravascular infusion of AAV9 vectors achieves widespread gene delivery to the CNS of mice16 and large animals.17,18 Therefore, systemic delivery of an AAV9-hIFNb vector may be effective for generating a widely distributed CNS network to combat the invasive and migratory properties of GBM. Here we tested this concept in an orthotopic xenograft model of invasive human GBM.

mL of cell suspension was injected stereotaxically into the left striatum. The stereotaxic coordinates for tumor implantation from bregma were (in mm): AP: +0.5, ML: 2.0 (left) and DV from brain surface: 22.5. Bilateral tumors were generated by injecting 50 000 GBM8-Fluc cells into both striata.

AAV Vector Design, Production, and Delivery All recombinant AAV9s used in the study were selfcomplementary (sc) vectors. scAAV9/CB-hIFNb and scAAV9/ CB-hIFNb -miRBS-1-122 vectors encode human interferon-b under the chicken b-actin promoter and cytomegalovirus enhancer (CB promoter) and carry a rabbit beta-globin polyadenylation (RBGpA) signal. The scAAV9/CB-hIFNb-miRBS-1-122 vector carries 3 copies of miR-1 and miR-122 binding sites (miRBS) in the 3 ′ untranslated region as described.20 The scAAV9/TBG-hIFNb vector carries a thyroxin-binding globulin (TBG) promoter to drive liver-specific gene expression.21 The scAAV9/CB-EGFP and scAAV9/TBG-EGFP vectors encode enhanced green fluorescence protein (EGFP). AAV9 vectors were produced at the University of Massachusetts Medical School Gene Therapy Center Viral Vector Core as described.22 Vector titers were determined by quantitative PCR (qPCR) of vector genomes using the following primers and probe specific for RBGpA (Eurofins):

Materials and Methods Fig. 2. Treatment by systemic delivery of scAAV9/CB-hIFNb before the onset Primer1: of rapid tumor growth is critical for long-term ′ survival benefit. (A) ; 5′ -GCCAAAAATTATGGGGACAT-3 Tumor-associated bioluminescence signal (TABS) is represented as fold change over signal at 1 week after tumor implantation. The timing of ′ ′ -ATTCCAACACACTATTGCAATG-3 ; Primer2: 5 Cell Culture treatment (with 3 ×1011 gc scAAV9/CB-IFNb) after tumor implantation is identified as follows: second week¼red, third week¼purple, Probe: 6FAM-ATGAAGCCCCTTGAGCATCTGACTTCT-TAMRA three-and-a-half fourth week¼green (treated), and PBS control group¼black (injected at second week). Data are shown as GBM8 cells wereweeks¼orange; a kind gift from Dr. Samuel Rabkin (Massachumean+SD. Arrows of corresponding colors indicate the times treatment For for each group.administration, (B) Kaplan-MeierAAV9 survival curveswere showing effectvia of systemic vectors injected setts General Hospital, Boston, Massachusetts). Theseofcells time oftransduced treatment onwith survival. curvesvector are shown for the same the groups thea same color coding) as mL for in TABS time. tail (with vein in total volume of 200 PBS.change In theover intracrawere theSurvival lentivirus CSCW2-Fluc14 ***P , .001. (C) Images of TABS in representative mice for each treatment time-point. Log-rank P values: ****P , .0001; Red circles (gc) are the copies of to generate GBM8-Fluc cells con- nial treatment paradigm, 7.6 × 109 genome IRES-mCherry as described regions of interest (ROI) for signal quantification. (D) Quantification TABS from imaging of live untreated GBM8-Fluc-bearing mice at treatment stitutively expressing firefly luciferase. Cells were grown as scAAV9/CB-hIFNb vector were infused in 2 mL at 200 nL/min time points after tumor implantation. Each data point on the plot is the TABS value from one mouse. Values are shown with group mean neurospheres in neurobasal media (21103-049, Gibco) supple- in the same stereotaxic coordinates used for tumor implanta(black horizontal bar) and range (bars with different colors). Unpaired 2-tailed t test results: ** P , .01. (E) Representative images of untreated mented with 3 mM L-glutamine (25-005-CI, Mediatech), 1x tion. For control groups, an equal volume of PBS was injected GBM8-Fluc-bearing mouse brains immunostained for human nuclei at treatment time points. Abbreviations: n, number of animals in the the mice all of the experiments. B27 (17504-044, Gibco), 0.5xtumor N2 supplement group;supplement ns, not significant; Str, left striatum (where was implanted);into W, weeks after for tumor implantation. (17502-048, Gibco), 2 mg/mL heparin (H3400, Sigma-Aldrich), 1x antibiotic-antimycotic solution (30-004-CI, Mediatech) and 1x amphotericin B (30-003-CF, Peripherally produced hIFNbMediatech), contributes20tong/mL the recombinant human bFGF (100-18B, PeproTech), and therapeutic effect of systemically delivered 20 ng/mL of recombinant human EGF (AF-100-15, PeproTech).

scAAV9/CB-IFNb

Because AAV9 transduces peripheral organs at high efficiency Animals after vascular delivery20,23 and the ubiquitous nature of the CB promoter used inmale scAAV9/CB-INFb, it is were possible that Sixto eight week-old athymic nude mice obtained peripherally expressed hIFNb contributes to the therapeutic from the National Cancer Institute for this study. All animal effect intracranial GBM8-Fluc tumors. Two AAV vectors studiesagainst were approved by the University of Massachusetts Medwere used Institutional to investigate this contribution: scAAV9/ ical School Animal Care and Use(1) Committee CB-hIFNb-miRBS-1-122 vectorGuide where following guidelines set (de-targeted) forth by the NIH’s forCB-driven the Care transgene de-targeted from liver, muscle, and and Use ofexpression Laboratorywas Animals (https://grants.nih.gov/grants/ 19 heart by insertion of miR-1 and miR-122 binding sites into olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf). 20 the transgene cassette as described and (2) scAAV9/TBGhIFNb (TBG) vector where transgene expression was driven by Orthotopic Xenografting the liver-specific thyroxin binding globulin (TBG) promoter.21 The efficacy of theseinto AAV9 wasmedium compared Two therapeutic days prior to implantation thevectors mice, the of with scAAV9/CB-hIFNb (CB vector) GBM8-Fluc tumor-bearing GBM8-Fluc cells was replaced withinfresh medium. On the day mice treatedGBM8-Fluc by systemiccells delivery. miR-1 and miR-122 reguof injection, wereThe dissociated into a single cell ′ lation of AAV-CB-hIFNb-miRBS-1-122 transgene expression was suspension by pipetting. Cells were washed twice in Dulbecco s validated first in cell culture Fig. S3). Accordingly, phosphate-buffered saline (Supplementary (PBS; 14190-250, Gibco) and resusplasma in concentration IFNb was significantly in mice pended the same to of a concentration of 50 000lower cells/mL. One 1512 2 of 11 Neuro-Oncology

Live Bioluminescence Imaging

treated with scAAV9/CB-hIFNb-miRBS-1-122 compared with Imaging of tumor-associated bioluminescence signal (TABS) scAAV9/CB-hIFNb (Fig. 4A). Also, as expected, vascular infusion was performed using the Xenogen IVIS 100 imaging system of 3× 1011 gc of scAAV9/TBG-EGFP vector did not result in appre(PerkinElmer) 3 minutes after intraperitoneal administration ciable EGFP expression in brain (Fig. 3A). Both bioluminescence of D-luciferin (4.5 mg). Image analysis was performed using imaging and survival (Fig. 4B and C) showed that treatment Living Image software (PerkinElmer). with de-targeted scAAV9/CB-hIFNb-miRBS-1-122 vector was less effective than scAAV9/CB-hIFNb as it required 1× 1012 gc/ Preparation of Tissue DNA and RNA and Quantification mouse to achieve comparable suppression of tumor growth of hIFNb Transcripts andVector 100% Genomes long-term and survival. Vector genome copy analysis in brain, DNA liver, was and skeletal muscle the 2 Blood vectors&toTissue have Total extracted usingshowed the DNeasy comparable biodistribution/transduction profiles (Supplemenkit (QIAGEN). DNA was diluted to a final concentration of tary 100 Fig. ng/mLfor S2). Therefore �3-fold lower therapeutic efficacy 50– vectorthe genome quantification by qPCR using of de-targeted vector couldand be probe. due to differences in peripheral RBGpolyA specific primers tissue expression hIFNb or a difference in brain expression Tissue RNA wasofisolated using TRIzol (15596-018, Invitrobecause nonspecificRNA miRNA interaction or both. Quantitative gen) andofDirect-zol MiniPrep (R2052, Zymo Research RT-PCR analysisRNA of hIFNb mRNA levels brain showed comparaCorporation). was treated with in TURBO DNase (AM1907, ble levelsfor in mice treatedatwith scAAV9/CB-hIFNb and scAAV9/ Ambion) 30 minutes 378C prior to reverse transcription CB-hIFNb-miRBS-1-122 vectors (Fig. However hIFNbBiosysmRNA using High Capacity RNA-to-cDNA kit4D). (4387406, Applied levels were significantly in liver andwith skeletal of tems). Quantitative PCRreduced was performed the muscle following mice treated with scAAV9/CB-hIFNb-miRBS-1-122 compared primers and probe for hIFNb (IDT): Primer-1: 5 ′ -GCAATTGAA ′ with those infused with the same dose of scAAV9/CB-hIFNb′ ; ; Primer-2: 5 ′ -TCATAGATGGTCAATGCGGC-3 TGGGAGGCTTG-3 ′ (Fig. 4E5and F). These results suggest that peripheral hIFNb Probe: -/6-FAM/TGTCAAAGT/ZEN/TCATCCTGTCCTTGAGGC/3IA Neuro-Oncology 5 of 11


BkFQ. Mouse HPRT1 expression was used as an internal reference gene to normalize all values (Assay ID: Mm00446968_m1; Applied Biosystems). Mean expression values for PBS group animals were considered to be background noise and thus were subtracted from all values.

Quantification of Human IFNb in Cell-conditioned Media and Mouse Plasma hIFNb in conditioned growth medium and mouse plasma was measured using ELISA assays (41 410 and 41 415, respectively; PBL Assay Science). Conditioned growth media were collected for ELISA 48 hours after transfection. Mouse plasma was collected 1 month after AAV infusion. Media and plasma were diluted 1:10 and 1:4,000 in PBS, respectively, for ELISA measurements.

Histological Analysis Brain cryosections (20 mm) and paraffin sections (5 mm) were used for histological studies. The primary antibodies used for immunohistochemistry were rabbit monoclonal anti-GFP (1:1000, G10362, Invitrogen) and rabbit monoclonal anti-Olig2 (1:250, ab109186, Abcam). Biotinylated goat anti-rabbit IgG (1:1000, BA-1000, Vector Laboratories) was used as secondary antibody. VECTASTAIN Elite ABC Kit (PK-6100, Vector Laboratories) and DAB substrate kit (SK-4100, Vector Laboratories) were used for immunohistochemical detection and Mayer′ s hematoxylin (Sigma) as counterstain. Cryosections (20 mm) were used to detect human GBM8-Fluc cells in the brain by immunofluorescence using mouse anti-human nuclei (1:150, MAB4383, EMD Millipore) and detection with Alexa-fluor 594 goat anti-mouse (1: 2000, A-11020, Invitrogen). Sections were also stained with 4′ ,6-diamidino-2-phenylindole (DAPI) (PI-62247, Thermo Fisher Scientific).

weight. In the 2 top doses of scAAV9/CB-hIFNb, TABS decreased over time and was indistinguishable from baseline (week-1 signal) by 2 weeks after treatment (4 weeks after tumor implantation). This remained unchanged over time (Fig. 1A and B). Treatment with 1 × 1011 gc showed partial response. TABS decreased to baseline levels in 2 mice while increasing over time in 3 mice, albeit with different kinetics than in the PBS control group (Fig. 1B). The scAAV9/CB-hIFNb dose response was also reflected in long-term survival with 100%, 88.89%, and 20% of animals alive at 244 days post tumor implantation in 1 × 1012, 3 × 1011, and 1 ×1011 gc dose groups, respectively (Fig. 1C). All animals from the PBS control group reached the humane endpoint between 42 and 49 days with a median survival of 46 days after tumor implantation. Histological analysis of the brains from animals that survived to 244 days revealed normal histology with no microscopic evidence of tumors (Fig. 1D, Supplementary Fig. S1). Interestingly, in animals that succumbed to disease progression in the 1 × 1011 gc group, there were large tumors with sharply defined borders to normal tissue that were composed of numerous well-defined tumorlets (arrows in Fig. 1D). This is in contrast to the diffuse nature of GBM8 tumors in mouse brain found in the PBS control group (Fig. 1D).

Therapeutic Outcome Is Dependent on Tumor Growth Phase at the Time of Treatment

To assess the effect of tumor growth phase on the therapeutic efficacy 3 ×10 11 gc (minimum effective dose) of scAAV9/ CB-hIFNb was infused at 2, 3, 3.5 (�84 hours after week-3 treatment), and 4 weeks after GBM8-Fluc tumor implantation. Treatment at week 2 and week 3 prevented tumor growth as suggested by TABS assessment over time (Fig. 2A). More importantly, this ultimately resulted in long-term survival of 100% and 88.9% of mice to 244 days after tumor implantation (Fig. 2B). Treatment at 3.5 and 4 weeks had a modest imGraphs and Statistical Analysis pact on TABS increase over time (Fig. 2A). Nonetheless, it Kaplan-Meier survival curves were plotted in Prism 6 (GraphPad resulted in significant increases in median survival to 60 and Software), and the Mantel-Cox log-rank test was used for stat- 61 days, respectively, compared with 46 days for PBS control istical analysis. Bar graphs were also plotted using the same mice. The maximum survivals were increased to 150 and software. And an unpaired 2-tailed t test was used for statisti- 80 days, respectively, compared with 54 days for PBS controls cal analysis. Calculated P values were defined as the probability (Fig. 2B). of null hypothesis being true; *P , .05; **P , .01; ***P , .001; Analysis of tumor burden by TABS at the treatment time ****P , .0001; ns, not significant (P . .05). points revealed that tumors remained largely unchanged between 2 andupon 3 but increased thereafter (Fig. 2Cvector and D). Fig. 3. Astrocytes and vascular endothelial cells are the predominant brain cellsweeks transduced vascular delivery of scAAV9-EGFP in Immunostaining of tumor sections for human nuclear antigen glioblastoma (GBM) tumor-bearing mice (A) Representative pictures of immunohistochemical staining for EGFP-expressing cells in brain Results 11 sections from GBM8-Fluc-bearing mice 3 weeks after tail vein delivery of and 3 ×10 gc/mouse of scAAV9/CB-EGFP Control GBM-specific OLIG2 marker at and the scAAV9/TBG-EGFP. time of treatment cormice were infused with PBS. 3,3′ -diaminobenzidine (DAB) was used for detection of this EGFP-positive cells (brown) the brain and hematoxylin roborated observation (Fig. 2E,in Supplementary Fig S2). Dose-dependent Therapeutic Response With Systemic was used for (B) High magnification pictures of 4 different brainresults areas (identified A by boxedbetween small case letters) from mice These suggest aincorrelation therapeutic efficaDelivery of counterstaining. scAAV9/CB-hIFNb injected with scAAV9/CB-EGFP vector. Large arrows in Ba and Bc indicatecy EGFP-expressing cells with astrocyte morphology. Small arrows in Bb of scAAV9/CB-hIFNb and tumor growth phase. To the therapeutic effectiveness of systemically andassess Bd indicate EGFP-positive cells with vasculature morphology.delivImages from one animal representative of each group (n ¼ 3). ered scAAV9/CB-hIFNb, athymic nude mice were treated with different vector doses (1 × 1011, 3 × 1011, and 1 × 1012 gc) or Systemically Delivered AAV9 Primarily Transduces expression contributed substantially toimplantation. the therapeutic efficacy PBS 2 weeks after GBM8-Fluc tumor We mea- scAAV9/TBG-IFNb was comparable to the other vectors (SuppleAstrocytes and Endothelial Cells in the of scAAV9/CB-hIFNb. This conclusion is supported by the sured TABS by live bioluminescence imaging to gain some in- mentary Fig. S4), but increased hIFNb mRNA levels were found Mouse Brain therapeutic results with liver-specific scAAV9/TBG-IFNb which Glioblastoma-bearing primarily in liver (Fig. 4D and F). Nonetheless, there was detectsight into changes in tumor growth kinetics in theinmouse both changed the of GBM8-Fluc able hIFNb mRNA present in the brains of mice treated brain doses after tested AAV treatment. In kinetics PBS-injected control tumor mice, Tumor-bearing mice were injected systemically with 3 ×with 10111× gc 12 growth (Fig. 4B)exponentially and improveduntil survival significantly (Fig. the 4C),hual- of 10 scAAV9/CB-EGFP gc of scAAV9/TBG-IFNb, at .2-log levelstranscomTABS increased the animals reached vector toalbeit determine the lower cell types beit lessendpoint effectively than scAAV9/CB-IFNb. biodistribution of duced pared with the other vectors (Fig. 4D). mane defined by .15% loss The in maximum body and thus were2likely mediators of the therapeutic effect. Neuro-Oncology1513 Neuro-Oncology 6 of 11 Neuro-Oncology 3 of 11



Materials and Methods Cell Culture GBM8 cells were a kind gift from Dr. Samuel Rabkin (Massachusetts General Hospital, Boston, Massachusetts). These cells were transduced with the lentivirus vector CSCW2-FlucIRES-mCherry as described14 to generate GBM8-Fluc cells constitutively expressing firefly luciferase. Cells were grown as neurospheres in neurobasal media (21103-049, Gibco) supplemented with 3 mM L-glutamine (25-005-CI, Mediatech), 1x B27 supplement (17504-044, Gibco), 0.5x N2 supplement (17502-048, Gibco), 2 mg/mL heparin (H3400, Sigma-Aldrich), 1x antibiotic-antimycotic solution (30-004-CI, Mediatech) and 1x amphotericin B (30-003-CF, Mediatech), 20 ng/mL recombinant human bFGF (100-18B, PeproTech), and 20 ng/mL of recombinant human EGF (AF-100-15, PeproTech).

Animals


AAV Vector Design, Production, and Delivery All recombinant AAV9s used in the study were selfcomplementary (sc) vectors. scAAV9/CB-hIFNb and scAAV9/ CB-hIFNb -miRBS-1-122 vectors encode human interferon-b under the chicken b-actin promoter and cytomegalovirus enhancer (CB promoter) and carry a rabbit beta-globin polyadenylation (RBGpA) signal. The scAAV9/CB-hIFNb-miRBS-1-122 vector carries 3 copies of miR-1 and miR-122 binding sites (miRBS) in the 3 ′ untranslated region as described.20 The scAAV9/TBG-hIFNb vector carries a thyroxin-binding globulin (TBG) promoter to drive liver-specific gene expression.21 The scAAV9/CB-EGFP and scAAV9/TBG-EGFP vectors encode enhanced green fluorescence protein (EGFP). AAV9 vectors were produced at the University of Massachusetts Medical School Gene Therapy Center Viral Vector Core as described.22 Vector titers were determined by quantitative PCR (qPCR) of vector genomes using the following primers and probe specific for RBGpA (Eurofins): Primer1: 5′ -GCCAAAAATTATGGGGACAT-3′ ; Primer2: 5′ -ATTCCAACACACTATTGCAATG-3′ ; Probe: 6FAM-ATGAAGCCCCTTGAGCATCTGACTTCT-TAMRA For systemic administration, AAV9 vectors were injected via the tail vein in a total volume of 200 mL in PBS. In the intracranial treatment paradigm, 7.6 × 109 genome copies (gc) of scAAV9/CB-hIFNb vector were infused in 2 mL at 200 nL/min in the same stereotaxic coordinates used for tumor implantation. For control groups, an equal volume of PBS was injected into the mice for all of the experiments.

Live Bioluminescence Imaging Imaging of tumor-associated bioluminescence signal (TABS) was performed using the Xenogen IVIS 100 imaging system (PerkinElmer) 3 minutes after intraperitoneal administration of D-luciferin (4.5 mg). Image analysis was performed using Living Image software (PerkinElmer).

Fig. 4. Peripheral expression of hIFNb contributes to the therapeutic effect of systemically infused scAAV9/CB-hIFNb in the GBM8 mouse model. (A) Sixto eight week-old male athymic nude mice were obtained scAAV9/CB-hIFNb (CB) or scAAV9/CB-hIFNb-miRBS-1-122 (de-targeted) vectors were infused at the DNA doses and shown or phosphate-buffered saline Preparation of Tissue RNA and Quantification from the National Institute forand thisplasma study.hIFNb All animal (PBS) at 2 weeks afterCancer tumor implantation levels measured 1 month later by ELISA (n ¼ 4 per group). Columns represent of Vector Genomes and hIFNb Transcripts studies were and approved by the University of , Massachusetts Medmean values, error bars indicate SD. ****P .0001 in unpaired 2-tailed t test. (B) Tumor-associated bioluminescence signal (TABS) kinetics ical School Institutional Animal Care at and Use after Committee Total DNA extracted usingCBthe DNeasy Blood & Tissue are represented as fold change over signal 1 week tumor implantation for was all treatment groups. vector, de-targeted vector, or following guidelines setvector) forth by theinjected NIH’s Guide forafter the Care scAAV9/TBG-hIFNb (TBG were 2 weeks tumor implantation at DNA doseswas shown. Datato area shown as mean+SD. (C) kit (QIAGEN). diluted final concentration of and Use of Laboratory Animals (https://grants.nih.gov/grants/ Kaplan-Meier survival curves for different treatment groups. In log-rank test, , ng/mLfor .01; ns, notvector significant (P . .05). (D– F) Comparison of hIFNb 50–**P 100 genome quantification by qPCR using 19 mRNA levels in brain, liver, and skeletal muscle in mice from different treatment groups at the humane or probe. study endpoints by RT-qPCR (n ¼ 3 olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf). RBGpolyA specific primers and per group). Top panels show the comparative delta CT values, and bottom panels expression. Data are shown as mean+SD. In Tissueshow RNA relative was isolated using TRIzol (15596-018, Invitrounpaired 2 tailed t test, *P , .05; **P , .01; ***P , .001; Abbreviation: ns, not significant (P . .05). gen) and Direct-zol RNA MiniPrep (R2052, Zymo Research

Orthotopic Xenografting

Two days prior to implantation into the mice, the medium of Systemic Delivery of scAAV9-hIFNb Is More Effective Than GBM8-Fluc cells was replaced with fresh medium. On the day Local Administration of Multifocal GBM cell of injection, GBM8-Fluc for cellsTreatment were dissociated into a single ′ We have previously shown thatwere intracranial of AAVrh8suspension by pipetting. Cells washed injection twice in Dulbecco s IFNb vectors leads tosaline complete ofGibco) established nonphosphate-buffered (PBS; regression 14190-250, and resus14 migratory orthotopic U87 brain tumors. HereOne we pended in the same to human a concentration of 50 000 cells/mL. 1514 2 of 11 Neuro-Oncology

Corporation). RNA was treated with TURBO DNase (AM1907, Ambion) for 30 minutes at 378C prior to reverse transcription sought to compare the effectiveness of local versus systemic using High Capacity RNA-to-cDNA kit (4387406, Applied BiosysdeliveryQuantitative of scAAV9/CB-hIFNb mice with bilateral tems). PCR wasinperformed with theGBM8-Fluc following ′ tumors to Mice5were treated primers andmimic probemultifocal for hIFNb recurrence. (IDT): Primer-1: -GCAATTGAA ′ ′ either by unilateral intracranial5 injection or systemic adminis-′ ; ; Primer-2: -TCATAGATGGTCAATGCGGC-3 TGGGAGGCTTG-3 tration 5of′ -/6-FAM/TGTCAAAGT/ZEN/TCATCCTGTCCTTGAGGC/3IA scAAV9/CB-IFNb. Probe: Neuro-Oncology 7 of 11


BkFQ. Bioluminescence Mouse HPRT1imaging expression showed was(Fig. used 5A as andan B) internal that tureference mors grew uninhibited gene to on normalize both sides of allthevalues brain in (Assay the untreatID: Mm00446968_m1; ed control group (Fig. Applied 5A). The Biosystems). average right-to-left Mean expression (R:L) TABS values ratioforfor PBSthis group group animals remained were considered at 1.05+0.11 to be background over time noise (Fig. 5B). andInthus the were groupsubtracted treated with from unilateral all values. intracranial injection, TABS decreased over time in the left (treated) hemisphere but increased rapidly in the untreated right hemisphere Quantification of reflected Human IFNb Cell-conditioned Media (Fig. 5A). This was in theinincrease in the right-to-left and Mouse Plasma TABS ratio over time (Fig. 5B). In contrast, TABS decreased over time in both hemispheres and eventually became undehIFNb in conditioned growth medium and mouse plasma was tectable inusing mice ELISA treated systemically and respectively; the average measured assays (41 410(Fig. and5A), 41 415, right-to-left TABS ratio remained unchanged at 1.04+0.11. The PBL Assay Science). Conditioned growth media were collected impact on48 survival withMouse the tumor-growth imfor ELISA hours was afterconsistent transfection. plasma was colaging data. The after median mice and treated by unilateral lected 1 month AAVsurvival infusion.ofMedia plasma were diintracranial of scAAV9/CB-IFNb was 56 days comluted 1:10 injection and 1:4,000 in PBS, respectively, for ELISA pared with 43 days for untreated animals, but nonetheless all measurements. animals in this cohort succumbed to tumor growth. In contrast, all animals in the systemic treatment group survived until the Histological Analysis endpoint (Fig. 5C). 8-month experimental Histological analysis of brains at endpoint (Fig. (5 5D) showed Brain cryosections (20 mm) and paraffin sections mm) were no evidence of residual tumorsThe in mice treated systemically. In used for histological studies. primary antibodies used for mice treated with unilateral intracranial injection, there were immunohistochemistry were rabbit monoclonal anti-GFP large tumor masses in the right hemisphere, but (1:1000, G10362, Invitrogen) and (untreated) rabbit monoclonal anti-Olig2 these had sharply defined borders (1:250, ab109186, Abcam).tumor-brain Biotinylatedparenchyma goat anti-rabbit IgG unlike the untreated tumors. This indicates scAAV9/ (1:1000, BA-1000, Vector Laboratories) was usedthat as secondary CB-IFNb injection in the leftABC hemisphere effectively antibody. VECTASTAIN Elite Kit (PK-6100, Vector prevented Laboratoipsilateral tumor growth,kit but it was insufficient to prevent distal ries) and DAB substrate (SK-4100, Vector Laboratories) were ′ tumorfor growth in the contralateral hemisphere. Nonetheless it used immunohistochemical detection and Mayer s hemaappears(Sigma) that local changed the phenotype the toxylin as treatment counterstain. Cryosections (20 mm)ofwere untreated tumor in theGBM8-Fluc contralateral used to detect human cellshemisphere. in the brain by immunoFinally, the presence of remnant GBM8-FLuc nuclei tumor cells was fluorescence using mouse anti-human (1:150, assessed in brain sections by immunostaining for oligodendroMAB4383, EMD Millipore) and detection with Alexa-fluor 594 cyte transcription as this is a protein highly goat anti-mouse factor-2 (1: 2000,(OLIG2) A-11020, Invitrogen). Sections expressed in glioblastoma stem cells.24,25 No OLIG2-positive were also stained with 4′ ,6-diamidino-2-phenylindole (DAPI) cells were apparent in theScientific). brains of systemically treated mice, (PI-62247, Thermo Fisher while the OLIG2 immunostaining patterns in the other 2 groups were consistent with the histological findings (Supplementary Graphs Fig. S5). and Statistical Analysis The present study curves showswere that plotted systemic scAAV9/CB-hIFNb Kaplan-Meier survival in Prism 6 (GraphPad gene therapy is an effective log-rank approach towas overcoming the Software), and the Mantel-Cox test used for statinvasiveness long-distance migratory properties of istical analysis.and Bar graphs were also plotted using the same glioblastoma. software. And an unpaired 2-tailed t test was used for statistical analysis. Calculated P values were defined as the probability of null hypothesis being true; *P , .05; **P , .01; ***P , .001; ****P , .0001; ns, not significant (P . .05). Discussion Our group has previously shown that local AAV-IFNb gene therResults apy is an effective approach for treating orthotopic human U87 GBM tumors in mice.14 However, in patients, unlike the noninDose-dependent Therapeutic Response With Systemic vasive U87 model, GBM cells migrate extensively in the brain. Delivery of scAAV9/CB-hIFNb Thus, reaching widely distributed cells may be challenging using intraparenchymal of AAV vectors evendelivwith To assess the therapeuticinjections effectiveness of systemically convection-enhanced delivery. Here wemice have were successfully ered scAAV9/CB-hIFNb, athymic nude treateddemwith 11 12 onstratedvector that systemic AAV9-IFNb different doses (1 delivery × 1011, 3of × 10 , and 1can × 10engineer gc) or brain to after form GBM8-Fluc a global tumor inhibitory network, PBS 2 cells weeks tumor implantation. We which meacan treat an invasive GBM model. while suredeffectively TABS by live bioluminescence imaging toBesides, gain some in14 our earlier showed prevention new tumor establishsight into study changes in tumor growth of kinetics in the mouse ment the contralateral hemisphere in a pretreatment brain in after AAV treatment. In PBS-injected controlmodel, mice, it was unsuccessful for treating established contralateral TABS increased exponentially until the animals reached the hutumors. In the current study, we show first timebody that mane endpoint defined by .15% lossforinthe maximum

systemic can be therapeutically effective fordecreased such disweight. Indelivery the 2 top doses of scAAV9/CB-hIFNb, TABS tant or multifocal The limitation of intraparenchymal over time and was tumors. indistinguishable from baseline (week-1 siginfusion AAV after vectors for treating widespread GBM implantumors nal) by 2of weeks treatment (4 weeks after tumor was alsoThis apparent here, albeit in the of an tation). remained unchanged overcontext time (Fig. 1Aartificial and B). 11 model of concurrent bilateral tumors. Theseresponse. results are consisgc showed partial TABS deTreatment with 1 × 10 tent withtoabaseline recent study injection an creased levels in in which 2 miceintracranial while increasing overof time AAVrh8-sTRAIL extended survival ofinmice withcontrol GBM8 in 3 mice, albeitvector with different kinetics than the PBS tumors, but1B). all animals ultimately succumbed to tumor growth group (Fig. in ,100 days.26 The scAAV9/CB-hIFNb dose response was also reflected in long-term survival with 100%, and injection 20% of animals Interestingly, although distal88.89%, intracranial was in12 alive at 244 days post tumor implantation 1 × 10 , 3× sufficient to achieve regression of establishedinGBM8 tumors, 11 11 10 it modified the histological phenotype of the invasive to , and 1 ×10 gc dose groups, respectively (Fig. 1C).GBM All anone offrom a noninvasive tumorgroup (Fig. reached 5D and E). is evident imals the PBS control theThis humane endfrom the distinct42 border between tumor and nontupoint between andnoticeable 49 days with a median survival of mordays tissues contrast to the diffuse pattern seen in the 46 after in tumor implantation. untreated control group. similar on tumor phenotype Histological analysis of Athe brainseffect from animals that survived was documented in normal mice treated systemically with the low to 244 days revealed histology with no microscopic evdose (1of ×tumors 1011 gc)(Fig. idence 1D, Supplementary Fig.1D). S1). Presently Interestingly, of scAAV9/CB-hIFNb (Fig. the in animals that succumbed to diseaseswitch progression the 1 × mechanism of low-level IFNb-induced from a in migratory gc group, there were phenotype large tumors with sharply 1011 to a local rapidly dividing is unknown, but defined further borders may to normal that were composed of numerous studies revealtissue new targets in the molecular networks – 29 well-defined (arrows Fig. 1D).27This is in contrast that regulate tumorlets GBM migration andininvasion. A previous therapeutic a systo the diffuse study naturereported of GBM8some tumors in mouseeffect brain of found in temically delivered AAV8 the PBS control group (Fig.vector 1D). encoding IFNb in U87 tumors grown in the flank of mice;30 however, flank GBM xenografts do not reproduce the challenges of brain gene delivery systemiTherapeutic Outcome Is Dependent cally or otherwise. To our knowledge, on thisTumor is the Growth first study showing of AAV9-IFNb induces complete Phase atthat thesystemic Time ofdelivery Treatment regression of tumors in a highly invasive orthotopic glioblastoTo assess the effect of tumor growth phase on the therapeutic ma model. efficacy 3 ×10 11 gc (minimum effective dose) of scAAV9/ We have used an immune-compromised athymic mouse CB-hIFNb was infused at 2, 3, 3.5 (�84 hours after week-3 model, which lacks T cells, for this study. Moreover, as human treatment), and 4 weeks after GBM8-Fluc tumor implantation. IFNb does not interact with the mouse IFNb receptors,31 it is Treatment at week 2 and week 3 prevented tumor growth as unlikely that mouse innate immune cells (such as dendritic suggested by TABS assessment over time (Fig. 2A). More imcells, macrophages, or NK cells) would be induced by human portantly, this ultimately resulted in long-term32survival of IFNb as was also suggested in a previous study. Therefore, 100% and 88.9% of mice to 244 days after tumor implantathe therapeutic effect documented here is likely due to a direct tion (Fig. 2B). Treatment at 3.5 and 4 weeks had a modest imeffect of IFNb on GBM8 tumor cells. In an immune-competent pact on TABS increase over time (Fig. 2A). Nonetheless, it organism, the immune stimulatory effect4,33,34 of speciesresulted in significant increases in median survival to 60 and matched IFNb would likely potentiate its antitumor role further. 61 days, respectively, compared with 46 days for PBS control In addition, one possibility is that IFNb gene therapy could be mice. The maximum survivals were increased to 150 and used as a concomitant therapy with temozolomide because it 80 days, respectively, compared with 54 days for PBS controls may help to increase the sensitivity of chemo-resistant tumor (Fig. 2B). cells as shown in anaplastic astrocytoma patients35 and glioma Analysis of tumor burden by TABS at the treatment time stem cells.36 IFNb gene therapy may also enhance the effect of points revealed that tumors remained largely unchanged beother therapeutic approaches such as antibodies targeting ontween weeks 2 and 3 but increased thereafter (Fig. 2C and D). cogenic receptors, similar to what was shown in a recent study Immunostaining of tumor sections10for human nuclear antigen with an antibody-IFNb conjugate. and GBM-specific OLIG2 marker at the time of treatment corIn this study, we observed that gene transfer efficiency roborated this observation (Fig. 2E, Supplementary Fig S2). in the tumor or surrounding striatum was noticeably low These results suggest a correlation between therapeutic efficacompared with cortex or periventricular region. Nonetheless, cy of scAAV9/CB-hIFNb and tumor growth phase. it appears that expression of IFNb from normal cells in the brain, and to some extent from peripheral tissues, contributed to the therapeutic The therapeutic effect docuSystemically Delivered benefit. AAV9 Primarily Transduces mented in this study lends support to the notion of exploring Astrocytes and Endothelial Cells in the systemic AAV gene therapy encoding secretory proteins with Glioblastoma-bearing Mouse Brain alpha, tumor necrosis antitumor properties (eg, interferon 11 factor alpha, TNF-related apoptosis-inducing ligand) Tumor-bearing mice were injected systemically with 3 × 10given gc that widespread direct cell transduction may be of scAAV9/CB-EGFP vectortumor to determine the cell types transchallenging. duced and thus were likely mediators of the therapeutic effect.





Animals


AAV Vector Design, Production, and Delivery All recombinant AAV9s used in the study were selfcomplementary (sc) vectors. scAAV9/CB-hIFNb and scAAV9/ CB-hIFNb -miRBS-1-122 vectors encode human interferon-b under the chicken b-actin promoter and cytomegalovirus enhancer (CB promoter) and carry a rabbit beta-globin polyadenylation (RBGpA) signal. The scAAV9/CB-hIFNb-miRBS-1-122 vector carries 3 copies of miR-1 and miR-122 binding sites (miRBS) in the 3 ′ untranslated region as described.20 The scAAV9/TBG-hIFNb vector carries a thyroxin-binding globulin (TBG) promoter to drive liver-specific gene expression.21 The scAAV9/CB-EGFP and scAAV9/TBG-EGFP vectors encode enhanced green fluorescence protein (EGFP). AAV9 vectors were produced at the University of Massachusetts Medical School Gene Therapy Center Viral Vector Core as described.22 Vector titers were determined by quantitative PCR (qPCR) of vector genomes using the following primers and probe specific for RBGpA (Eurofins): Primer1: 5′ -GCCAAAAATTATGGGGACAT-3′ ; Primer2: 5′ -ATTCCAACACACTATTGCAATG-3′ ; Probe: 6FAM-ATGAAGCCCCTTGAGCATCTGACTTCT-TAMRA For systemic administration, AAV9 vectors were injected via the tail vein in a total volume of 200 mL in PBS. In the intracranial treatment paradigm, 7.6 × 109 genome copies (gc) of scAAV9/CB-hIFNb vector were infused in 2 mL at 200 nL/min in the same stereotaxic coordinates used for tumor implantation. For control groups, an equal volume of PBS was injected into the mice for all of the experiments.


Six- to eight week-old male athymic nude mice were obtained from the National Cancer Institute for this study. All animal Preparation of Tissue DNA and RNA and Quantification studies were approved by the University of Massachusetts Med- of Vector Genomes and hIFNb Transcripts ical School Institutional Animal Care and Use Committee Total DNA was extracted using the DNeasy Blood & Tissue following guidelines set forth by the NIH’s Guide for the Care kit (QIAGEN). DNA was diluted to a final concentration of and Use of Laboratory Animals (https://grants.nih.gov/grants/ 50– 100 ng/mLfor vector genome quantification by qPCR using 19 intraparenchymal administration for treatment of multifocal glioblastoma. Fig. 5. Systemic delivery of scAAV9-hIFNb vector is more effective than olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf). RBGpolyA specific primers and probe. (A) Images of tumor-associated bioluminescence signal (TABS) over time (weeks 2 – 6) in representative mice with bilateral GBM8-Fluc tumors Tissue RNA was isolated using TRIzol (15596-018, Invitro11 treated by systemic (3×10 gc) or unilateral (left tumor) intracranial (7.6×109gc) administration of scAAV9/CB-hIFNb vector. Separate regions gen) and Direct-zol RNA MiniPrep (R2052, Zymo Research of interest (ROIs; red boxes) over the left and right side of the head were used to quantify TABS for each bilateral tumor. (B) Change in right-to-left Orthotopic Xenografting Corporation). RNA was treated withline) TURBO DNase intracranial (AM1907, hemisphere (R:/L) TABS ratio from week 1 to week 6 after tumor implantation in mice treated by systemic (green or unilateral Two days prior to implantation into the mice, the medium of Ambion) for 30 minutes at 378C prior to reverse transcription injection (red line), and untreated controls (black line). All values are normalized to the week-1 signal (n ¼ 5 mice per group) (C) Kaplan-Meier GBM8-Fluc cells fresh(green medium. Onunilateral the day(leftusing High Capacity(red RNA-to-cDNA Applied Biosyssurvival curves for was mice replaced treated bywith systemic line) or tumor) intracranial line) deliverykit of (4387406, scAAV9/CB-hIFNb vector and of injection, GBM8-Fluc cells line) were(ndissociated a single cell tems).pictures Quantitative wasstained performed with the following untreated control group (black ¼ 5 mice perinto group). (D) Representative of brain PCR sections with hematoxylin and eosin ′ suspension pipetting. Cells were (untreated: washed twice s primers and probe for hIFNb 5 ′ -GCAATTGAA from differentbygroups at the humane day in 43Dulbecco and unilateral intracranial treatment: day 56) or(IDT): study Primer-1: endpoint (day 244). On the ′ ′ ′ left, most panel wholesaline brain sections are shown Gibco) at low and magnification. On the right 2 panels, high magnification images are shown of the ; Primer-2: 5 -TCATAGATGGTCAATGCGGC-3 ; phosphate-buffered (PBS; 14190-250, resus- TGGGAGGCTTG-3 ′ corresponding areas indicated in the whole brain sections by L1, L2, or L3 and R1, R2, or R3. Scale bars represent 100 mm. pended in the same to a concentration of 50 000 cells/mL. One Probe: 5 -/6-FAM/TGTCAAAGT/ZEN/TCATCCTGTCCTTGAGGC/3IA 1516 2 of 11 Neuro-Oncology

Neuro-Oncology 9 of 11


BkFQ. Mouse HPRT1 Material expression was used as an internal Supplementary reference gene to normalize all values (Assay ID: Supplementary material is available online at Neuro-Oncology Mm00446968_m1; Applied Biosystems). Mean expression val(http://neuro-oncology.oxfordjournals.org/). ues for PBS group animals were considered to be background noise and thus were subtracted from all values.

Funding Quantification of Human IFNb in Cell-conditioned Media This work was Plasma supported in part by grant R01NS066310 (M.S-E.) from and Mouse the NIH.

hIFNb in conditioned growth medium and mouse plasma was measured using ELISA assays (41 410 and 41 415, respectively; PBL Assay Science). Conditioned growth media were collected Conflict of interest statement. G.G. is a founder of Voyager Therapeutics for ELISA 48 hours after transfection. Mouse plasma was coland holds equity inafter the company. G.G. is an inventor patentswere licensed lected 1 month AAV infusion. Media andon plasma dito Voyager Therapeutics. luted 1:10 and 1:4,000 in PBS, respectively, for ELISA measurements.

References

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healthy Interferon Cytokine Res. 2000;20(10): weight. In thevolunteers. 2 top dosesJ of scAAV9/CB-hIFNb, TABS decreased 857– 866. over time and was indistinguishable from baseline (week-1 signal) 2 weeks after DH, treatment after tumor 14. by Maguire CA, Meijer LeRoy SG,(4etweeks al. Preventing growthimplanof brain tation). Thisbyremained over time (Fig. 2008;16(10): 1A and B). tumors creating aunchanged zone of resistance. Mol Ther. 1695– 1702. Treatment with 1 × 1011 gc showed partial response. TABS decreased to baseline levels 2 mice increasing over time 15. Wakimoto H, Kesari S, in Farrell CJ, while et al. Human glioblastomain 3 mice, albeit with different kinetics than in PBS control derived cancer stem cells: establishment of the invasive glioma groupmodels (Fig. 1B). and treatment with oncolytic herpes simplex virus vectors. Cancer Res. 2009;69(8):3472–3481. The scAAV9/CB-hIFNb dose response was also reflected in long-term survival 100%, 88.89%, and A, 20% animals 16. Foust KD, Nurre E,with Montgomery CL, Hernandez ChanofCM, Kaspar 12 aliveBK. at Intravascular 244 days post implantation in 1 × 10neurons , 3× AAV9tumor preferentially targets neonatal 11 1011,and andadult 1 ×10 gc dose respectively (Fig.–65. 1C). All anastrocytes. Natgroups, Biotechnol. 2009;27(1):59 imals fromS,the PBS control group the humane end17. Duque Joussemet B, Riviere C, etreached al. Intravenous administration pointofbetween 42 and 49 days withtransgene a median survival of self-complementary AAV9 enables delivery to adult 46 days after tumor implantation. motor neurons. Mol Ther. 2009;17(7):1187– 1196. the brains from animals thatdelivery survived 18. Histological Bevan AK, analysis Duque S, of Foust KD, et al. Systemic gene in to 244 days revealed withbrain, no microscopic evlarge species for normal targetinghistology spinal cord, and peripheral idence of tumors 1D, Supplementary S1). Interestingly, tissues for (Fig. pediatric disorders. MolFig.Ther. 2011;19(11): in animals that succumbed to disease progression in the 1 × 1971– 1980. 11 gc group, for there large tumors with defined 10 19. Committee the were Update of the Guide for thesharply Care and Use of borders to normal tissue thatforwere composed Laboratory Animals. Guide the Care and Useof of numerous Laboratory well-defined (arrows inDC: Fig.National 1D). This is in contrast Animals. tumorlets 8th ed. Washington, Academies Press; to the diffuse nature of GBM8 tumors in mouse brain found in 2011. the PBS control group (Fig. 1D). 20. Xie J, Xie Q, Zhang H, et al. MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Mol Ther. 2011;19(3):526– 535.

Therapeutic Outcome Is Dependent on Tumor Growth 21. Yan Z, Yan H, Ou H. Human thyroxine binding globulin (TBG) Phase at the directs Time of Treatment promoter efficient and sustaining transgene expression in liver-specific Gene. 2012;506(2):289– 294. To assess the effectpattern. of tumor growth phase on the therapeutic 11 gcF,(minimum scAAV9/ efficacy ×10 22. Ayuso3E, Mingozzi Montane J, eteffective al. High AAVdose) vector of purity results CB-hIFNb was infused 2, 3, 3.5 (�84 hours after week-3 in serotypeand attissue-independent enhancement of transduction Gene GBM8-Fluc Ther. 2010;17(4):503–510. treatment), and efficiency. 4 weeks after tumor implantation. Treatment atHe week 2 andXC, week prevented tumor to growth as 23. Chen BD, CH, Chen et al.3Targeting transgene the heart suggested bywith TABS assessment time (Fig. More imand liver AAV9 by differentover promoters. Clin2A). Exp Pharmacol portantly, ultimately resulted Physiol.this 2015;42(10):1108– 1117. in long-term survival of 100% and 88.9% of mice to 244 days tumor implanta24. Ligon KL, Alberta JA, Kho AT, et al. Theafter oligodendroglial lineage tion (Fig. 2B).OLIG2 Treatment at 3.5 and 4 weeks in had a modest immarker is universally expressed diffuse gliomas. pact Jon TABS increase over time (Fig. 2A). Neuropathol Exp Neurol. 2004;63(5):499– 509.Nonetheless, it resulted in significant survival of toOLIG2 60 and 25. Trepant AL, Bouchartincreases C, Rorive S,inetmedian al. Identification as 61 days, respectively, compared with 46 days for PBS control the most specific glioblastoma stem cell marker starting from mice.comparative The maximum were increased to microarray 150 and analysissurvivals of data from similar DNA chip 80 days, respectively, compared with 54 days platforms. Tumour Biol. 2015;36(3):1943– 1953.for PBS controls (Fig. 26. 2B). Crommentuijn MH, Maguire CA, Niers JM, et al. Intracranial Analysis of tumor burden TABS at Cthe treatment time AAV-sTRAIL combined withby lanatoside prolongs survival in points tumors remained unchanged beanrevealed orthotopicthat xenograft mouse model largely of invasive glioblastoma. tween weeks and 3 but increased thereafter (Fig. 2C and D). Mol Oncol.22016;10(4):625–634. Immunostaining of tumor sections for human nuclear antigen 27. Godlewski J, Bronisz A, Nowicki MO, Chiocca EA, Lawler S. and GBM-specific marker atswitch the time of treatment microRNA-451:OLIG2 A conditional controlling glioma corcell roborated this observation (Fig. 2E, Supplementary S2). proliferation and migration. Cell Cycle. 2010;9(14):2742–Fig 2748. These results suggest a correlation between therapeutic effica28. Williams SP, Nowicki MO, Liu F, et al. Indirubins decrease glioma cy ofinvasion scAAV9/CB-hIFNb and tumor growth phase. by blocking migratory phenotypes in both the tumor and stromal endothelial cell compartments. Cancer Res. 2011; 71(16):5374– 5380.

Systemically Delivered AAV9 Primarily Transduces 29. Nevo I, Woolard K, Cam M, et al. Identification of molecular Astrocytes and Endothelial Cells in the pathways facilitating glioma cell invasion in situ. PLoS One. Glioblastoma-bearing 2014;9(11):e111783. Mouse Brain Tumor-bearing were with 3efficacy × 1011 gc 30. Streck CJ, mice Dickson PV,injected Ng CY, systemically et al. Antitumor of AAV-mediated systemic of interferon-beta. Cancer Gene of scAAV9/CB-EGFP vector delivery to determine the cell types transTher. –106.mediators of the therapeutic effect. duced and2006;13(1):99 thus were likely



31. Harari D, Abramovich ZozulyaPreviously, A, et al. Bridging the shown species a widespread therapeuticR, effect. we have divide:expression transgenicof mice humanized for from type-Ia interferon that local human IFNb (hIFNb) recombiPLoS One. 2014;9(1):e84259. nant response. adeno-associated viral (AAV) vector successfully inhibits 32. Qinof XQ, Beckham C,human Brown JL, Lukashev Barsoum J. Human growth intracranial U87 tumorsM, through gene deliv14 mouse IFN-beta exhibits U87therapy brain tumors growdifferent rapidly ery toand normal cells in the brain.gene mechanisms in mouse models. Mol Ther. 2001;4(4): in theanti-tumor mouse brain, but mostly as large spheroids with distinct 356–364. borders, and thus do not reproduce the invasiveness and 33. Goodbourn S, Didcock L, Randall Interferons:for cell signalling, migratory properties of GBM that areRE. responsible recurrence. immune modulation, antiviral response and virus countermeasures. To test the effectiveness of AAV-hIFNb gene therapy in a more J Gen Virol. 2000;81(Pt 10):2341–2364. realistic model, we carried out studies using GBM8 cells, which upon transplantation into mouse brain produce diffuse tumors with extensive infiltration and long-distance migration.15 A potential approach to counteract the properties of GBM cells that limits the efficacy of local interventions is to generate a disperse network of endogenous cells expressing IFNb to prevent single tumor cell infiltration and growth (ie, matching the delivery modality to the disease characteristics). Intravascular infusion of AAV9 vectors achieves widespread gene delivery to the CNS of mice16 and large animals.17,18 Therefore, systemic delivery of an AAV9-hIFNb vector may be effective for generating a widely distributed CNS network to combat the invasive and migratory properties of GBM. Here we tested this concept in an orthotopic xenograft model of invasive human GBM.


Animals Six- to eight week-old male athymic nude mice were obtained from the National Cancer Institute for this study. All animal studies were approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee following guidelines set forth by the NIH’s Guide for the Care and Use of Laboratory Animals (https://grants.nih.gov/grants/ olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf).19

Orthotopic Xenografting Two days prior to implantation into the mice, the medium of GBM8-Fluc cells was replaced with fresh medium. On the day of injection, GBM8-Fluc cells were dissociated into a single cell suspension by pipetting. Cells were washed twice in Dulbecco′ s phosphate-buffered saline (PBS; 14190-250, Gibco) and resuspended in the same to a concentration of 50 000 cells/mL. One 1518 2 of 11 Neuro-Oncology

34. ofStark Kerr IM, Williams BR, Silverman RH, Schreiber mL cell GR, suspension was injected stereotaxically into RD. theHow left cells The respond to interferons. Annufor Revtumor Biochem. 1998;67: striatum. stereotaxic coordinates implantation 264. were (in mm): AP: +0.5, ML: 2.0 (left) and DV from227– bregma 35. Fujimaki T, Ishii H,22.5. Matsuno A, Arai H, Nakagomi T. Effectiveness of from brain surface: Bilateral tumors were generated by interferon-beta and temozolomide therapy against injecting 50 000 GBM8-Fluc cells intocombination both striata. temozolomide-refractory recurrent anaplastic astrocytoma. World J Surg Oncol. 2007;5:89.

AAV Vector Design, Production, and Delivery

36.

Shen D, Guo CC, Wang J, et al. Interferon-alpha/beta enhances

All recombinant AAV9sagainst used MGMT-positive in the study were selftemozolomide activity glioma stem-like complementary (sc) vectors. scAAV9/CB-hIFNb and scAAV9/ cells. Oncol Rep. 2015;34(5):2715– 2721. CB-hIFNb -miRBS-1-122 vectors encode human interferon-b under the chicken b-actin promoter and cytomegalovirus enhancer (CB promoter) and carry a rabbit beta-globin polyadenylation (RBGpA) signal. The scAAV9/CB-hIFNb-miRBS-1-122 vector carries 3 copies of miR-1 and miR-122 binding sites (miRBS) in the 3 ′ untranslated region as described.20 The scAAV9/TBG-hIFNb vector carries a thyroxin-binding globulin (TBG) promoter to drive liver-specific gene expression.21 The scAAV9/CB-EGFP and scAAV9/TBG-EGFP vectors encode enhanced green fluorescence protein (EGFP). AAV9 vectors were produced at the University of Massachusetts Medical School Gene Therapy Center Viral Vector Core as described.22 Vector titers were determined by quantitative PCR (qPCR) of vector genomes using the following primers and probe specific for RBGpA (Eurofins): Primer1: 5′ -GCCAAAAATTATGGGGACAT-3′ ; Primer2: 5′ -ATTCCAACACACTATTGCAATG-3′ ; Probe: 6FAM-ATGAAGCCCCTTGAGCATCTGACTTCT-TAMRA For systemic administration, AAV9 vectors were injected via the tail vein in a total volume of 200 mL in PBS. In the intracranial treatment paradigm, 7.6 × 109 genome copies (gc) of scAAV9/CB-hIFNb vector were infused in 2 mL at 200 nL/min in the same stereotaxic coordinates used for tumor implantation. For control groups, an equal volume of PBS was injected into the mice for all of the experiments.


Preparation of Tissue DNA and RNA and Quantification of Vector Genomes and hIFNb Transcripts Total DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN). DNA was diluted to a final concentration of 50– 100 ng/mLfor vector genome quantification by qPCR using RBGpolyA specific primers and probe. Tissue RNA was isolated using TRIzol (15596-018, Invitrogen) and Direct-zol RNA MiniPrep (R2052, Zymo Research Corporation). RNA was treated with TURBO DNase (AM1907, Ambion) for 30 minutes at 378C prior to reverse transcription using High Capacity RNA-to-cDNA kit (4387406, Applied Biosystems). Quantitative PCR was performed with the following primers and probe for hIFNb (IDT): Primer-1: 5 ′ -GCAATTGAA TGGGAGGCTTG-3 ′ ; Primer-2: 5 ′ -TCATAGATGGTCAATGCGGC-3 ′ ; Probe: 5 ′ -/6-FAM/TGTCAAAGT/ZEN/TCATCCTGTCCTTGAGGC/3IA Neuro-Oncology 11 of 11

Highly efficient retinal gene delivery with helper-dependent adenoviral vectors.

Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

Dextranation of bioreducible cationic polyamide for systemic gene delivery.

Nanoparticle-Mediated Target Delivery of TRAIL as Gene Therapy for Glioblastoma.

Selective lentiviral gene delivery to CD133-expressing human glioblastoma stem cells.

Sui generis: gene therapy and delivery systems for the treatment of glioblastoma.

Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex.

Experimental Curative Fluorescence-guided Surgery of Highly Invasive Glioblastoma Multiforme Selectively Labeled With a Killer-reporter Adenovirus.

Systemic manifestations of invasive amebiasis.

Delivery of hypoxia and glioma dual-specific suicide gene using dexamethasone conjugated polyethylenimine for glioblastoma-specific gene therapy.

Broad functional correction of molecular impairments by systemic delivery of scAAVrh74-hSGSH gene delivery in MPS IIIA mice.

Virus-Inspired Self-Assembled Nanofibers with Aggregation-Induced Emission for Highly Efficient and Visible Gene Delivery.

Molecular replacement: tricks and treats.

Patterned substrates of nano-graphene oxide mediating highly localized and efficient gene delivery.

Oligopeptide-terminated poly(β-amino ester)s for highly efficient gene delivery and intracellular localization.

Enhancing dendritic cell-based vaccination for highly aggressive glioblastoma.

Non-invasive glioblastoma immunoprofiling by printed peptide arrays.

Systematic review of protein biomarkers of invasive behavior in glioblastoma.

Safety and angiogenic effects of systemic gene delivery of a modified erythropoietin.

GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

Rapid screening of gene function by systemic delivery of morpholino oligonucleotides to live mouse embryos.

Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA.

Highly effective inhibition of lung cancer growth and metastasis by systemic delivery of siRNA via multimodal mesoporous silica-based nanocarrier.

Convection-enhanced delivery for the treatment of glioblastoma.