AAV-mediated gene therapy restores natural fertility and improves physical function in the Lhcgr-deficient mouse model of Leydig cell failure

Abstract

Leydig cell failure (LCF) caused by gene mutations leads to testosterone deficiency, infertility and reduced physical function. Adeno-associated virus serotype 8 (AAV8)-mediated gene therapy shows potential in treating LCF in the Lhcgr-deficient (Lhcgr^–/–) mouse model. However, the gene-treated mice still cannot naturally sire offspring, indicating the modestly restored testosterone and spermatogenesis in AAV8-treated mice remain insufficient to support natural fertility. Recognizing this, we propose that enhancing gene delivery could yield superior results. Here, we screened a panel of AAV serotypes through in vivo transduction of mouse testes and identified AAVDJ as an impressively potent vector for testicular cells. Intratesticular injection of AAVDJ achieved markedly efficient transduction of Leydig cell progenitors, marking a considerable advance over conventional AAV8 vectors. AAVDJ-Lhcgr gene therapy was well tolerated and resulted in significant recovery of testosterone production, substantial improvement in sexual development, and remarkable restoration of spermatogenesis in Lhcgr^–/– mice. Notably, this therapy restored fertility in Lhcgr^–/– mice through natural mating, enabling the birth of second-generation. Additionally, this treatment led to remarkable improvements in adipose, muscle, and bone function in Lhcgr^–/– mice. Collectively, our findings underscore AAVDJ-mediated gene therapy as a promising strategy for LCF and suggest its broader potential in addressing various reproductive disorders.

References

[1] ZirkinBR, Papadopoulos V. Leydig cells: formation, function, and regulation. Biol Reprod. 2018; 99(1):101-111.

[2] TeerdsKJ, Huhtaniemi IT. Morphological and functional maturation of Leydig cells: from rodent models to primates. Hum Reprod Update. 2015; 21(3):310-328.

[3] LiL, Papadopoulos V. Advances in stem cell research for the treatment of primary hypogonadism. Nat Rev Urol. 2021; 18(8):487-507.

[4] GarzaS, Papadopoulos V. Testosterone recovery therapy targeting dysfunctional Leydig cells. Andrology. 2023; 11(5):816-825.

[5] MendoncaBB, CostaEM, BelgoroskyA, Rivarola MA, DomeniceS. 46,XY DSD due to impaired androgen production. Best Pract Res Clin Endocrinol Metab. 2010; 24(2):243-262.

[6] MakelaJA, Koskenniemi JJ, VirtanenHE, ToppariJ. Testis Development. Endocr Rev. 2019; 40(4):857-905.

[7] SaloniaA, Rastrelli G, HackettG, et al. Paediatric and adult-onset male hypogonadism. Nat Rev Dis Primers. 2019; 5(1):38.

[8] BhasinS, BritoJP, CunninghamGR, et al. Testosterone therapy in men with hypogonadism: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018; 103(5):1715-1744.

[9] KathrinsM, Niederberger C. Diagnosis and treatment of infertility-related male hormonal dysfunction. Nat Rev Urol. 2016; 13(6):309-323.

[10] DunbarCE, HighKA, JoungJK, Kohn DB, OzawaK, SadelainM. Gene therapy comes of age. Science. 2018; 359:6372.

[11] LiC, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet. 2020; 21(4):255-272.

[12] XiaK, WangF, LaiX, et al. AAV-mediated gene therapy produces fertile offspring in the Lhcgr-deficient mouse model of Leydig cell failure. Cell Rep Med. 2022; 3(11):100792.

[13] KellyDM, JonesTH. Testosterone and obesity. Obes Rev. 2015; 16(7):581-606.

[14] O’ConnellMD, Wu FC. Androgen effects on skeletal muscle: implications for the development and management of frailty. Asian J Androl. 2014; 16(2):203-212.

[15] FerlinA, SeliceR, CarraroU, Foresta C. Testicular function and bone metabolism—beyond testosterone. Nat Rev Endocrinol. 2013; 9(9):548-554.

[16] BasariaS. Male hypogonadism. Lancet. 2014; 383(9924):1250-1263.

[17] RaoCV, LeiZM. Consequences of targeted inactivation of LH receptors. Mol Cell Endocrinol. 2002; 187(1–2):57-67.

[18] LeiZM, MishraS, ZouW, et al. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Mol Endocrinol. 2001; 15(1):184-200.

[19] XiaK, MaY, FengX, et al. Endosialin defines human stem Leydig cells with regenerative potential. Hum Reprod. 2020; 35(10):2197-2212.

[20] XiaK, WangF, TanZ, et al. Precise correction of Lhcgr mutation in stem Leydig cells by prime editing rescues hereditary primary hypogonadism in mice. Adv Sci (Weinh). 2023; 10(29):e2300993.

[21] WatanabeS, Kanatsu-Shinohara M, OgonukiN, MatobaS, OguraA, ShinoharaT. In vivo genetic manipulation of Spermatogonial stem cells and their microenvironment by adeno-associated viruses. Stem Cell Rep. 2018; 10(5):1551-1564.

[22] WangZ, WuW, KimMS, Cai D. GnRH pulse frequency and irregularity play a role in male aging. Nat Aging. 2021; 1(10):904-918.

[23] ZhangY, KimMS, JiaB, et al. Hypothalamic stem cells control ageing speed partly through exosomal miRNAs. Nature. 2017; 548(7665):52-57.

[24] XiaoYZ, YangM, XiaoY, et al. Reducing hypothalamic stem cell senescence protects against aging-associated physiological decline. Cell Metab. 2020; 31(3):534-548.e5.

[25] IvellR, WadeJD, Anand-IvellR. INSL3 as a biomarker of Leydig cell functionality. Biol Reprod. 2013; 88(6):147.

[26] GrimmD, LeeJS, WangL, et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol. 2008; 82(12):5887-5911.

[27] SenisE, Mosteiro L, WilkeningS, et al. AAVvector-mediated in vivo reprogramming into pluripotency. Nat Commun. 2018; 9(1):2651.

[28] KumarSRP, Hoffman BE, TerhorstC, de JongYP, HerzogRW. The balance between CD8(+) T cell-mediated clearance of AAV-encoded antigen in the liver and tolerance is dependent on the vector dose. Mol Ther. 2017; 25(4):880-891.

[29] WangD, TaiPWL, GaoG. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019; 18(5):358-378.

[30] GuercioG, Costanzo M, GrinsponRP, ReyRA. Fertility issues in disorders of sex development. Endocrinol Metab Clin North Am. 2015; 44(4):867-881.

[31] NguyenGN, Everett JK, KafleS, et al. A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells. Nat Biotechnol. 2021; 39(1):47-55.

[32] WuFC, TajarA, BeynonJM, et al. Identification of late-onset hypogonadism in middle-aged and elderly men. N Engl J Med. 2010; 363(2):123-135.

RIGHTS & PERMISSIONS

2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.