High and prolonged sulfamidase secretion by the liver of MPS-IIIA mice following hydrodynamic tail vein delivery of antibiotic-free pFAR4 plasmid vector

Abstract

Mucopolysaccharidosis type IIIA (MPS-IIIA) or Sanfilippo A syndrome is a lysosomal storage genetic disease that results from the deficiency of the N-sulfoglucosamine sulfohydrolase (SGSH) protein, a sulfamidase required for the degradation of heparan sulfate glycosaminoglycans (GAGs). The accumulation of these macromolecules leads to somatic organ pathologies, severe neurodegeneration and death. To assess a novel gene therapy approach based on prolonged secretion of the missing enzyme by the liver, mediated by hydrodynamic gene delivery, we first compared a kanamycin and an antibiotic-free expression plasmid vector, called pFAR4. Thanks to the reduced vector size, pFAR4 derivatives containing either a ubiquitous or a liver-specific promoter mediated a higher reporter gene expression level than the control plasmid. Hydrodynamic delivery of SGSH-encoding pFAR4 into MPS-IIIA diseased mice led to high serum levels of sulfamidase protein that was efficiently taken up by neighboring organs, as shown by the correction of GAG accumulation. A similar reduction in GAG content was also observed in the brain, at early stages of the disease. Thus, this study contributes to the effort towards the development of novel biosafe non-viral gene vectors for therapeutic protein expression in the liver, and represents a first step towards an alternative gene therapy approach for the MPS-IIIA disease.

Cationic microbubbles and antibiotic-free miniplasmid for sustained ultrasound–mediated transgene expression in liver

“…In our study, we showed that a long term transgene expression could be obtained using pFAR4 miniplasmids. The advantages of these miniplasmids have been proven in vivo upon hydrodynamic injection thanks to their reduced size, nontoxic and non-immunogenic side effects [22,23]. These features may play a significant role in gene transfer efficiency by sonoporation.…”

“…Removal of all or part of bacterial sequences from gene vectors (e.g. in minicircles or in pFAR4 miniplasmids, respectively) allowed to alleviate this silencing effect [22,23]. Alternatively, the use of molecular tools (DNA transposons or integrases) that mediate transgene integration into host genome led, in mice, to high and prolonged therapeutic levels of Factor IX that is deficient in hemophilia B patients.…”

“…We chose a miniplasmid devoid of antibiotic resistance sequence, called pFAR4 [27]. The absence of this sequence leads to a small size plasmid which was shown to be highly efficient in transfecting cells in vitro [27] and in vivo [23]. Finally, the parameters of low-energy unfocused ultrasound were also optimized using image-guided delivery.…”

Neuronopathic Types of Mucopolysaccharidoses: Pathogenesis and Emerging Treatments

“…В некоторых исследованиях с использованием вну-тривенного введения аденоассоциированного (AAV8) и лентивирусного векторов, а также других способов доставки гена в клетки печени (гидродинамических инъ-екций плазмид; микроциркулярной ДНК, представля-ющей собой плазмиду, лишенную некоторых бактери-альных последовательностей ДНК; транспозона «спящая красавица», способного к интеграции в геном клеток хозяина) продемонстрирована возможность уменьше-ния степени выраженности патологических изменений не только в соматических клетках, но и в тканях головно-го мозга экспериментальных животных моделей МПС I, IIIА и VII [4,37,38]. По мнению некоторых исследовате-лей, механизм данного явления может быть связан с про-никновением через ГЭБ непрерывно синтезируемого на периферии фермента, преодолением вирусным векто-ром незрелого ГЭБ или несостоятельностью ГЭБ в связи с течением воспалительного процесса у используемых лабораторных животных [8,10].…”

Pre-clinical Mouse Models of Neurodegenerative Lysosomal Storage Diseases

“…et al, 2010), ERT(Lee et al, 2011(Lee et al, , 2014Higuchi et al, 2012;Hong et al, 2012;Sohn et al, 2018 Sohn et al, ) et al, 2007Haurigot et al, 2013), AI(Arfi et al, 2011), BMT(Lau et al, 2012),LV (McIntyre et al, 2008), SRT(Roberts et al, 2010) Mgat3 (D31N)(Bhattacharyya et al, 2001) + + AAV(Ruzo et al, 2012;Haurigot et al, 2013), ERT(Gustavsson et al, 2019), GT(Quiviger et al, 2014), SRT(Roberts et al, 2007) Mgat3(CKO) (Lau et al, 2017(Lau et al, ) et al, 2004Ribera et al, 2015), ERT(Kan et al, 2014),LV (Di Natale et al, 2005Blanz et al, 2008;Damme et al, 2011) NEU1 Sialidosis, Type I & II Neu1 (−/−) (de Geest et al, 2002) + < Neu1 (V54M) (Bonten et al, 2013) + + Chaperone-AAV (Bonten et al, 2013)(Continued)…”