Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases

Federica Esposito; Fabio Dell'Aquila; Manuel Rhiel; Stefano Auricchio; Kay Ole Chmielewski; Geoffroy Andrieux; Rita Ferla; Paula Sureda Horrach; Arjun Padmanabhan; Roberto Di Cunto; Simone Notaro; Manel Llado Santeularia; Melanie Boerries; Margherita Dell'Anno; Edoardo Nusco; Agnese Padula; Sofia Nutarelli; Tatjana I. Cornu; Nicolina Cristina Sorrentino; Pasquale Piccolo; Ivana Trapani; Toni Cathomen; Alberto Auricchio

doi:10.1016/j.xcrm.2024.101619

Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases

Federica Esposito, Fabio Dell'Aquila, Manuel Rhiel, Stefano Auricchio, Kay Ole Chmielewski, Geoffroy Andrieux, Rita Ferla, Paula Sureda Horrach, Arjun Padmanabhan, Roberto Di Cunto, Simone Notaro, Manel Llado Santeularia, Melanie Boerries, Margherita Dell'Anno, Edoardo Nusco, Agnese Padula, Sofia Nutarelli, Tatjana I. Cornu, Nicolina Cristina Sorrentino, Pasquale PiccoloIvana Trapani, Toni Cathomen, Alberto Auricchio

Research output: Contribution to journal › Article

Abstract

Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3 0 end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.

Original language	English
Pages (from-to)	1-14
Number of pages	14
Journal	Cell Reports Medicine
Volume	5
DOIs	https://doi.org/10.1016/j.xcrm.2024.101619
Publication status	Published - 2024
Externally published	Yes

Keywords

AAV
CAST-Seq
CRISPR-Cas9
HITI
genome editing
persistent transgene expression
homology-independent targeted integration
inherited diseases
in vivo
liver
mucopolysaccharidosis type VI
hemophilia A

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10.1016/j.xcrm.2024.101619

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Esposito, F., Dell'Aquila, F., Rhiel, M., Auricchio, S., Chmielewski, K. O., Andrieux, G., Ferla, R., Horrach, P. S., Padmanabhan, A., Di Cunto, R., Notaro, S., Santeularia, M. L., Boerries, M., Dell'Anno, M., Nusco, E., Padula, A., Nutarelli, S., Cornu, T. I., Sorrentino, N. C., ... Auricchio, A. (2024). Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases. Cell Reports Medicine, 5, 1-14. https://doi.org/10.1016/j.xcrm.2024.101619

@article{c01520d0412c4cf9af8290836f2f8990,

title = "Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases",

abstract = "Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3 0 end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.",

keywords = "AAV, CAST-Seq, CRISPR-Cas9, HITI, genome editing, persistent transgene expression, homology-independent targeted integration, inherited diseases, in vivo, liver, mucopolysaccharidosis type VI, hemophilia A, AAV, CAST-Seq, CRISPR-Cas9, HITI, genome editing, persistent transgene expression, homology-independent targeted integration, inherited diseases, in vivo, liver, mucopolysaccharidosis type VI, hemophilia A",

author = "Federica Esposito and Fabio Dell'Aquila and Manuel Rhiel and Stefano Auricchio and Chmielewski, {Kay Ole} and Geoffroy Andrieux and Rita Ferla and Horrach, {Paula Sureda} and Arjun Padmanabhan and {Di Cunto}, Roberto and Simone Notaro and Santeularia, {Manel Llado} and Melanie Boerries and Margherita Dell'Anno and Edoardo Nusco and Agnese Padula and Sofia Nutarelli and Cornu, {Tatjana I.} and Sorrentino, {Nicolina Cristina} and Pasquale Piccolo and Ivana Trapani and Toni Cathomen and Alberto Auricchio",

year = "2024",

doi = "10.1016/j.xcrm.2024.101619",

language = "English",

volume = "5",

pages = "1--14",

journal = "Cell Reports Medicine",

issn = "2666-3791",

publisher = "Cell Press",

}

Esposito, F, Dell'Aquila, F, Rhiel, M, Auricchio, S, Chmielewski, KO, Andrieux, G, Ferla, R, Horrach, PS, Padmanabhan, A, Di Cunto, R, Notaro, S, Santeularia, ML, Boerries, M, Dell'Anno, M, Nusco, E, Padula, A, Nutarelli, S, Cornu, TI, Sorrentino, NC, Piccolo, P, Trapani, I, Cathomen, T & Auricchio, A 2024, 'Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases', Cell Reports Medicine, vol. 5, pp. 1-14. https://doi.org/10.1016/j.xcrm.2024.101619

TY - JOUR

T1 - Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases

AU - Esposito, Federica

AU - Dell'Aquila, Fabio

AU - Rhiel, Manuel

AU - Auricchio, Stefano

AU - Chmielewski, Kay Ole

AU - Andrieux, Geoffroy

AU - Ferla, Rita

AU - Horrach, Paula Sureda

AU - Padmanabhan, Arjun

AU - Di Cunto, Roberto

AU - Notaro, Simone

AU - Santeularia, Manel Llado

AU - Boerries, Melanie

AU - Dell'Anno, Margherita

AU - Nusco, Edoardo

AU - Padula, Agnese

AU - Nutarelli, Sofia

AU - Cornu, Tatjana I.

AU - Sorrentino, Nicolina Cristina

AU - Piccolo, Pasquale

AU - Trapani, Ivana

AU - Cathomen, Toni

AU - Auricchio, Alberto

PY - 2024

Y1 - 2024

N2 - Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3 0 end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.

AB - Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3 0 end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.

KW - AAV

KW - CAST-Seq

KW - CRISPR-Cas9

KW - HITI

KW - genome editing

KW - persistent transgene expression

KW - homology-independent targeted integration

KW - inherited diseases

KW - in vivo

KW - liver

KW - mucopolysaccharidosis type VI

KW - hemophilia A

KW - AAV

KW - CAST-Seq

KW - CRISPR-Cas9

KW - HITI

KW - genome editing

KW - persistent transgene expression

KW - homology-independent targeted integration

KW - inherited diseases

KW - in vivo

KW - liver

KW - mucopolysaccharidosis type VI

KW - hemophilia A

UR - http://hdl.handle.net/10807/295816

U2 - 10.1016/j.xcrm.2024.101619

DO - 10.1016/j.xcrm.2024.101619

M3 - Article

SN - 2666-3791

VL - 5

SP - 1

EP - 14

JO - Cell Reports Medicine

JF - Cell Reports Medicine

ER -

Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases

Abstract

Keywords

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