Transgenic rescue of Krabbe disease in the twitcher mouse

Abstract

Dedicated with respect and appreciation to Professor Robert A Lazzarini, our teacher and mentor, who made all this research possibleThe twitcher mouse is a natural model of Krabbe disease caused by galactocerebrosidase (GALC) deficiency. Previous attempts at rescuing the twitcher mouse by bone marrow transplantion, viral transduction, or transgenesis were only partially successful. Here, we report the transgenic (tg) rescue of the twitcher mouse with a BAC clone harboring the entire GALC. The twi/twi/hGALC tg mice exhibited growth, motor function, and fertility similar to those of nonaffected animals. These animals had normal levels of GALC activity in brain and were free of the typical twitcher demyelinating pathology. Surprisingly, GALC expression in twi/twi hGALC tg kidneys was low and galactocerebroside storage was only partially cleared. Nonetheless, these mice have been maintained for over 1 year without any sign of disease. Since pathological damage associated with GALC deficiency is confined to the nervous system, our work represents the first successful rescue of the twitcher mouse and opens the possibility of developing novel therapeutic approaches.

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe’s disease in the Twitcher mouse

“…Recombinant adenoviruses and lentiviral vectors resulted in the efficient reconstitution of GALC expression in Twitcher neural cells, but did not limit the progression of the disease in vivo [34,35]. Transgenic rescue of the Twitcher mouse was also attempted using human GALC cDNA [36] and a BAC clone containing the entire human GALC gene [37]. Although transgenic Twitcher mice were rescued in the latter approach, this germ-line therapy can not be considered clinically relevant at present.…”

Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease

“…Neurological manifestations of the patients could be significantly improved following transplantation 35, 36. On the other hand, animal experiments that introduced the GALC gene into twitcher mice and cultured cells by virus vectors or transgenic technology showed various degrees of correction of biochemical, pathological and clinical phenotypes suggesting the potential usefulness of gene therapy strategy in GLD 19, 37–39. The previous virus‐mediated gene therapy studies in twitcher mice 19, 40 have provided important points: (i) Gene transfer must be initiated from an early stage of disease course.…”

Animal transgenesis: an overview

“…Similarly, the use of BAC/PAC transgenes expressing fluorescent protein reporters in the optically transparent zebrafish has provided unparalleled visualization of gene expression in a living organism (Yang et al 2006. BAC transgenic strategies have also been used to trace neural lineages (Placantonakis et al 2009), identify sites of synthesis of IL-7 (Repass et al 2009), create models of Parkinson's disease using a truncated mutant parkin (Lu et al 2009), and rescue the Krabbe disease phenotype of the twitcher mouse where cDNA transgenic approaches had proven unsuccessful (De Gasperi et al 2004). YACs while technically more difficult to work with have allowed transgenesis with extremely large genes such as the *400 kb amyloid precursor protein (APP) (Lamb et al 1993).…”