Modulation of the liver immune microenvironment by the adeno-associated virus serotype 8 gene therapy vector

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in both glial cells and neurons. MLD results from an inherited deficiency of arylsulfatase A (ARSA) and myelin degeneration in the central and peripheral nervous systems. Currently, no effective treatment is available for the most frequent late infantile (LI) form of MLD after symptom onset. The LI form results in rapid neurological degradation and early death. ARSA enzyme must be rapidly and efficiently delivered to brain and spinal cord oligodendrocytes of patients with LI MLD in order to potentially stop the progression of the disease. We previously showed that brain gene therapy with adeno-associated virus serotype rh10 (AAVrh10) driving the expression of human ARSA cDNA alleviated most long-term disease manifestations in MLD mice but was not sufficient in MLD patient to improve disease progression. Herein, we evaluated the short-term effects of intravenous AAVPHP.eB delivery driving the expression of human ARSA cDNA under the control of the cytomegalovirus/b-actin hybrid (CAG) promoter in 6-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 3 months, a single intravenous injection of AAVPHP.eB-hARSA-HA resulted in correction of brain and spinal cord sulfatide storage, and improvement of astrogliosis and microgliosis in brain and spinal cord of treated animals. These results strongly support to consider the use of AAVPHP.eB-hARSA vector for intravenous gene therapy in symptomatic rapidly progressing forms of MLD.

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Adeno-associated virus as a gene therapy vector: strategies to neutralize the neutralizing antibodies

Clinical and Experimental Medicine ◽

10.1007/s10238-019-00557-8 ◽

2019 ◽

Vol 19 (3) ◽

pp. 289-298 ◽

Cited By ~ 9

Author(s):

Majid Lotfinia ◽

Meghdad Abdollahpour-Alitappeh ◽

Behzad Hatami ◽

Mohammad Reza Zali ◽

Morteza Karimipoor

Keyword(s):

Gene Therapy ◽

Neutralizing Antibodies ◽

Adeno Associated Virus ◽

Gene Therapy Vector

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Clinical Evaluation of a Recombinant Adeno-Associated Virus (rAAV) Alpha-1 Antitrypsin (AAT) Gene Therapy Vector.

10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a2908 ◽

2009 ◽

Author(s):

J Chulay ◽

M Brantly ◽

L Wang ◽

C Mueller ◽

M Humphries ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Evaluation ◽

Adeno Associated Virus ◽

Gene Therapy Vector ◽

Alpha 1 Antitrypsin

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The 37/67-Kilodalton Laminin Receptor Is a Receptor for Adeno-Associated Virus Serotypes 8, 2, 3, and 9

Journal of Virology ◽

10.1128/jvi.00878-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9831-9836 ◽

Cited By ~ 264

Author(s):

Bassel Akache ◽

Dirk Grimm ◽

Kusum Pandey ◽

Stephen R. Yant ◽

Hui Xu ◽

...

Keyword(s):

Gene Therapy ◽

Cultured Cells ◽

Human Gene ◽

Laminin Receptor ◽

Transduction Efficiency ◽

Adeno Associated Virus ◽

Virus Serotype ◽

Tumor Gene

ABSTRACT Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.

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OneBac: Platform for Scalable and High-Titer Production of Adeno-Associated Virus Serotype 1–12 Vectors for Gene Therapy

Human Gene Therapy ◽

10.1089/hum.2013.184 ◽

2014 ◽

Vol 25 (3) ◽

pp. 212-222 ◽

Cited By ~ 71

Author(s):

Mario Mietzsch ◽

Sabrina Grasse ◽

Catherine Zurawski ◽

Stefan Weger ◽

Antonette Bennett ◽

...

Keyword(s):

Gene Therapy ◽

High Titer ◽

Adeno Associated Virus ◽

Virus Serotype ◽

Serotype 1

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Reduced Immunogenicity of Intraparenchymal Delivery of Adeno-Associated Virus Serotype 2 Vectors: Brief Overview

Current Gene Therapy ◽

10.2174/1566523221666210922155413 ◽

2021 ◽

Vol 21 ◽

Author(s):

Wuh-Liang Hwu ◽

Shin-Ichi Muramatsu ◽

Bruria Gidoni-Ben-Zeev

Keyword(s):

Gene Therapy ◽

Immune Response ◽

Clinical Trial Data ◽

Trial Data ◽

Acid Decarboxylase ◽

Adeno Associated Virus ◽

Amino Acid Decarboxylase ◽

Virus Serotype ◽

The Brain ◽

Decarboxylase Deficiency

: Preexisting immunity to adeno-associated virus (AAV) poses a concern in AAV vector–mediated gene therapy. Localized administration of low doses of carefully chosen AAV serotypes can mitigate the risk of an immune response. This article will illustrate the low risk of immune response to AAV serotype 2 vector–mediated gene therapy to the brain with support from clinical trial data in aromatic L-amino acid decarboxylase deficiency and Parkinson disease.

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Targeted Modifications in Adeno-Associated Virus Serotype (AAV)- 8 Capsid Improves Its Hepatic Gene Transfer Efficiency in Vivo

Blood ◽

10.1182/blood.v120.21.2045.2045 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2045-2045

Author(s):

Dwaipayan Sen ◽

Nishanth Gabriel ◽

Sathish Kumar Yesupatham ◽

Rekha Samuel ◽

Rupali A Gadkari ◽

...

Keyword(s):

Gene Therapy ◽

Conflicts Of Interest ◽

Innate Immune ◽

Hemophilia B ◽

Transduction Efficiency ◽

Egfp Expression ◽

Adeno Associated Virus ◽

Virus Serotype ◽

Antibody Levels

Abstract Abstract 2045 Recombinant adeno-associated virus vectors based on serotype (AAV)-8 have shown significant promise for liver directed gene therapy of hemophilia B. However, in a recent clinical trial, two patients who received highest dose (2×1012 vg/kg) of the self-complementary (sc)AAV8 vector developed capsid specific T cells that required glucocorticoid therapy to attenuate this response [Nathwani et al, New Eng J Med, 2011]. Thus, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery, we modified specific serine/threonine kinase or ubiquitination targets on AAV8 capsid to improve its transduction efficiency. To test this, point mutations at specific serine (S), threonine (T) or lysine (K) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant capsids were evaluated for their liver transduction efficiency at a dose of 5 × 1010 vgs/ animal in C57BL/6 mice in vivo. Two of the AAV8-S>A mutants (S279A and S501A) and a K137R mutant vector, demonstrated significantly higher EGFP expression (3.6 to 12.5 fold) in the liver compared to animals that received WT-AAV8 vectors alone (Figure 1). The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response [interleukin (IL)-6, IL-12, tumor necrosis factor α, Kupffer cells (KC) and innate immune responsive toll like receptors (TLR)-9] with a concomitant 2-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies also revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (22 fold), lungs (9.7 fold) and muscle (8.4 fold) tissue when compared to WT-AAV8 vectors. Further on-going studies with the optimal mutant scAAV8 vector expressing human coagulation factor IX in murine models of hemophilia B, will demonstrate the feasibility of the use of these novel vectors for potential gene therapy of hemophilia B. Figure 1: Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Figure 1:. Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Disclosures: No relevant conflicts of interest to declare.

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Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

Journal of Virology ◽

10.1128/jvi.03125-14 ◽

2014 ◽

Vol 89 (6) ◽

pp. 3038-3048 ◽

Cited By ~ 24

Author(s):

Lauriel F. Earley ◽

Yasuhiro Kawano ◽

Kei Adachi ◽

Xiao-Xin Sun ◽

Mu-Shui Dai ◽

...

Keyword(s):

Gene Therapy ◽

Amino Acid ◽

Intracellular Localization ◽

Viral Capsid ◽

Nucleolar Localization ◽

Capsid Assembly ◽

Dependent Manner ◽

Carboxy Terminus ◽

Adeno Associated Virus ◽

Virus Serotype

ABSTRACTAssembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2144–184). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and β-galactosidase–AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2144–184has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly.IMPORTANCEAdeno-associated virus (AAV) has become a popular and successful vector forin vivogene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a nonstructural, nucleolar-localizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for virion formation. Here, we identify clusters of basic amino acids in the carboxy terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.

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