Hair Cell Transduction Efficiency of Single- and Dual-AAV Serotypes in Adult Murine Cochleae

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Abstract

Gene delivery is a key component for the treatment of genetic hearing loss. To date, a myriad of adeno-associated virus (AAV) serotypes and surgical approaches have been employed to deliver transgenes to cochlear hair cells, but the efficacy of dual transduction remains unclear. Herein, we investigated cellular tropism of single injections of AAV serotype 1 (AAV1), AAV2, AAV8, AAV9, and Anc80L65, and quantitated dual-vector co-transduction rates following co-injection of AAV2 and AAV9 vectors in adult murine cochlea. We used the combined round window membrane and canal fenestration (RWM+CF) injection technique for vector delivery. Single AAV2 injections were most robust and transduced 96.7% ± 1.1% of inner hair cells (IHCs) and 83.9% ± 2.0% of outer hair cells (OHCs) throughout the cochlea without causing hearing impairment or hair cell loss. Dual AAV2 injection co-transduced 96.9% ± 1.7% of IHCs and 65.6% ± 8.95% of OHCs. Together, RWM+CF-injected single or dual AAV2 provides the highest auditory hair cell transduction efficiency of the AAV serotypes we studied. These findings broaden the application of cochlear gene therapy targeting hair cells.

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Abstract

Transduction efficiency of inner and outer hair cells in the mature murine cochlea is excellent with adeno-associated virus serotype 2 (AAV2) when a round window membrane and canal fenestration technique is used. Dual-vector co-transduction of AAV2:AVV2 is equally robust and efficient, making AAV2 an excellent choice for single and/or dual applications that require hair cell transduction.

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Emerging Issues in AAV-Mediated In Vivo Gene Therapy

Pasqualina Colella, Giuseppe Ronzitti, Federico Mingozzi (2017)

In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, has established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective “therapeutic products” is, however, a long process that involves several cycles of iterations from bench to bedside that are required to address issues encountered during drug development. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from preclinical and clinical studies.

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Quantitative analysis of the packaging capacity of recombinant adeno-associated virus.

J. Dong, P Fan, Margaret Frizzell (1996)

Recombinant adeno-associated viruses (AAV) are among the most promising vectors for gene therapy of genetic diseases, including cystic fibrosis (CF). However, because of its small genome size, the capacity of AAV to package a therapeutic gene is limited. The efficiency of packaging the cystic fibrosis transmembrane conductance Regulator (CFTR) gene into AAV will be an important factor in determining whether recombinant AAV can be developed as a vector for transferring CFTR cDNA to the airway epithelia of patients with CF. Current understanding of the AAV biology suggests that AAV can package a genome slightly larger than the size of a wild-type genome. The precise range of the genome size and the efficiency of packaging have not been defined. Using a series of AAV vectors with progressively-increasing genome size, we were able to analyze quantitatively the packaging efficiency in relation to the vector size and to determine the size limit for packaging. The packaging efficiencies of AAV vectors of variable sizes were determined directly by assaying DNA contents of viral particles, and indirectly by analyzing their efficiency in transfer of a chloramphenicol acetyltransferase (CAT) reporter gene into target cells. Our studies showed that the optimal size of AAV vector is between 4.1 and 4.9 kb. Although AAV can package a vector larger than its genome size, up to 5.2 kb, the packaging efficiencies in this large size range were sharply reduced. When the AAV genome size was smaller than 4.1 kb, the packaging efficiency was also suboptimal. In contrast, when the size of the genome was less than half the length of the wild-type genome, two copies of the vector were packaged into each virion, suggesting that the copy number control during packaging is a "head-full" mechanism. Because the length of the minimal cDNA of CFTR is about 4.5 kb, these results suggest it is possible to package the CFTR gene into AAV if the combined length of transcriptional elements and ITRs is kept under 500 bp. The results of this study are important for directing the design of AAV vectors for efficient gene transfer, as well as for a better understanding of the mechanism of AAV genome packaging.

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A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear

Lukas Landegger, Bifeng Pan, Charles Askew … (2017)

Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities 1, 2 . Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector 3 , for transgene delivery to the mouse cochlea. Cochlear explants incubated with Anc80L65 encoding eGFP demonstrated high level transduction of inner and outer hair cells (60–100%). Injection of Anc80L65 through the round window membrane resulted in highly efficient transduction of inner and outer hair cells, a substantial improvement over conventional adeno-associated virus (AAV) vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

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Author and article information

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Richard J.H. Smith

Journal

Journal ID (nlm-ta): Mol Ther Methods Clin Dev

Journal ID (iso-abbrev): Mol Ther Methods Clin Dev

Title: Molecular Therapy. Methods & Clinical Development

Publisher: American Society of Gene & Cell Therapy

ISSN (Electronic): 2329-0501

Publication date PMC-release: 13 May 2020

Publication date Collection: 12 June 2020

Publication date (Electronic): 13 May 2020

Volume: 17

Pages: 1167-1177

Affiliations

[1 ]Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa, 285 Newton Road, 5270 CBRB, Iowa City, IA 52242, USA

[2 ]Department of Otolaryngology–Head and Neck Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-Cho, Kita-Ku, Okayama 700-8558, Japan

[3 ]Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan

[4 ]Department of Otolaryngology–Head and Neck Surgery, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

[5 ]Grousbeck Gene Therapy Center, Ocular Genomics Institute, Schepens Eye Research Institute and Mass Eye and Ear, Boston, MA, USA

[6 ]The Broad Institute of Harvard and MIT, Cambridge, MA, USA

[7 ]Iowa Institute of Human Genetics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

Author notes

[∗ ]Corresponding author Richard J.H. Smith, MD, Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa, 285 Newton Road, 5270 CBRB, Iowa City, IA 52242, USA. richard-smith@ 123456uiowa.edu

[8]

These authors contributed equally to this work.

Article

Publisher ID: S2329-0501(20)30094-2

DOI: 10.1016/j.omtm.2020.05.007

PMC ID: 7270144

PubMed ID: 32518805

SO-VID: a90ae2ba-8a99-454f-9d0f-7c07487c1a1e

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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Hair Cell Transduction Efficiency of Single- and Dual-AAV Serotypes in Adult Murine Cochleae

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Hearing Loss and Restoration

Most cited references 33

Emerging Issues in AAV-Mediated In Vivo Gene Therapy

Quantitative analysis of the packaging capacity of recombinant adeno-associated virus.

A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear

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