Mouse Survival Motor Neuron Alleles That Mimic SMN2 Splicing and Are Inducible Rescue Embryonic Lethality Early in Development but Not Late

Abstract

Spinal muscular atrophy (SMA) is caused by low survival motor neuron (SMN) levels and patients represent a clinical spectrum due primarily to varying copies of the survival motor neuron-2 (SMN2) gene. Patient and animals studies show that disease severity is abrogated as SMN levels increase. Since therapies currently being pursued target the induction of SMN, it will be important to understand the dosage, timing and cellular requirements of SMN for disease etiology and potential therapeutic intervention. This requires new mouse models that can induce SMN temporally and/or spatially. Here we describe the generation of two hypomorphic Smn alleles, SmnC-T-Neo and Smn2B-Neo. These alleles mimic SMN2 exon 7 splicing, titre Smn levels and are inducible. They were specifically designed so that up to three independent lines of mice could be generated, herein we describe two. In a homozygous state each allele results in embryonic lethality. Analysis of these mutants indicates that greater than 5% of Smn protein is required for normal development. The severe hypomorphic nature of these alleles is caused by inclusion of a loxP-flanked neomycin gene selection cassette in Smn intron 7, which can be removed with Cre recombinase. In vitro and in vivo experiments demonstrate these as inducible Smn alleles. When combined with an inducible Cre mouse, embryonic lethality caused by low Smn levels can be rescued early in gestation but not late. This provides direct genetic evidence that a therapeutic window for SMN inductive therapies may exist. Importantly, these lines fill a void for inducible Smn alleles. They also provide a base from which to generate a large repertoire of SMA models of varying disease severities when combined with other Smn alleles or SMN2-containing mice.

SMN-targeted therapeutics for spinal muscular atrophy: are we SMArt enough yet?

“…In humans lacking SMN production from SMN1, SMN2 phenotypically modifies development (15,16). Several groups have also demonstrated that early postnatal induction of SMN is critical for prolonged survival in transgenic SMA mouse models (17,18). In order to address the precise temporal requirements for SMN in postnatal development, Kariya and colleagues developed mouse models in which SMN knockdown could be temporally induced (19).…”

Spinal Muscular Atrophy: Journeying From Bench to Bedside

“…Expectedly, SMN levels in 8-copy SMN2 Smn -/-mice were restored to near normal. In the years since this early work was reported, the strategy of exploiting the human SMN2 gene has been modified in subtle ways to generate a number of additional SMA model mice [62][63][64][65][66]. Their use, as described below, has not only led to novel insights into the cellular and molecular basis of the human disease, but has also served as a springboard for the design and development of promising therapies for the patient population.…”

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

“…Due to the dedicated efforts of several groups, there is a diversity of available mouse models to test drug candidates (see Table 5 for details). These mouse models of SMA cover a wide range of pathological phenotypes from very severe mice that survive only a few days after birth, 27 to those that exhibit overt progressive motor circuit and neuromuscular pathology, 28,219,220 to intermediate SMA models, 42,221,222 and to very mildly affected animals. 26,41,49 A variety of compounds with different modes of action have been tested in mouse models of SMA, and the protocols for both SMN mRNA and protein expression as well as phenotypic readouts (e.g., survival, motor circuit structure and function, muscle mass, strength, and peripheral tissue necrosis) are well documented.…”