A novel humanized mouse model of Huntington disease for preclinical development of therapeutics targeting mutant huntingtin alleles.

Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global benefit. Thus there is a need for preclinical models of HD recapitulating human HTT genetics. We previously generated a humanized mouse model of HD, Hu97/18, by intercrossing BACHD and YAC18 mice with knockout of the endogenous mouse HD homolog (Hdh). Hu97/18 mice recapitulate the genetics of HD, having two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of Caucasian descent. We have now generated a companion model, Hu128/21, by intercrossing YAC128 and BAC21 mice on the Hdh-/- background. Hu128/21 mice have two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1, and for preclinical screening of personalized HTT lowering therapies in HD patients of East Asian descent.

Dose and Radioadaptive Response Analysis of Micronucleus Induction in Mouse Bone Marrow.

Enhanced cellular DNA repair efficiency and suppression of genomic instability have been proposed as mechanisms underlying radio-adaptive responses following low-dose radiation exposures. We previously showed that low-dose γ irradiation does not generate radio-adaptation by lowering radiation-induced cytogenetic damage in mouse spleen. Since radiation may exert tissue-specific effects, we extended these results here by examining the effects of γ radiation on cytogenetic damage and proliferative index in bone marrow erythrocytes of C57BL/6 and BALB/c mice. In C57BL/6 mice, the induction of micronuclei in polychromatic erythrocytes (MN-PCE) was observed at radiation doses of 100 mGy and greater, and suppression of erythroblast maturation occurred at doses of >500 mGy. A linear dose-response relationship for MN-PCE frequencies in C57BL/6 mice was established for radiation doses between 100 mGy and 1 Gy, with departure from linearity at doses of >1 Gy. BALB/c mice exhibited increased MN-PCE frequencies above baseline following a 20 mGy radiation exposure but did not exhibit radio-sensitivity relative to C57BL/6 mice following 2 Gy exposure. Radio-adaptation of bone marrow erythrocytes was not observed in either strain of mice exposed to low-dose priming γ irradiation (single doses of 20 mGy or 100 mGy or multiple 20 mGy doses) administered at various times prior to acute 2 Gy irradiation, confirming the lack of radio-adaptive response for induction of cytogenetic damage or suppression or erythrocyte proliferation/maturation in bone marrow of these mouse strains.

An enhanced Q175 knock-in mouse model of Huntington disease with higher mutant huntingtin levels and accelerated disease phenotypes.

Huntington disease (HD) model mice with heterozygous knock-in (KI) of an expanded CAG tract in exon 1 of the mouse huntingtin (Htt) gene homolog genetically recapitulate the mutation that causes HD, and might be favoured for preclinical studies. However, historically these mice have failed to phenotypically recapitulate the human disease. Thus, homozygous KI mice, which lack wildtype Htt, and are much less relevant to human HD, have been used. The zQ175 model was the first KI mouse to exhibit significant HD-like phenotypes when heterozygous. In an effort to exacerbate HD-like phenotypes and enhance preclinical utility, we have backcrossed zQ175 mice to FVB/N, a strain highly susceptible to neurodegeneration. These Q175F mice display significant HD-like phenotypes along with sudden early death from fatal seizures. The zQ175 KI allele retains a floxed neomycin resistance cassette upstream of the Htt gene locus and produces dramatically reduced mutant Htt as compared to the endogenous wildtype Htt allele. By intercrossing with mice expressing cre in germ line cells, we have excised the neo cassette from Q175F mice generating a new line, Q175FΔneo (Q175FDN). Removal of the neo cassette resulted in a ∼2 fold increase in mutant Htt and rescue of fatal seizures, indicating that the early death phenotype of Q175F mice is caused by Htt deficiency rather than by mutant Htt. Additionally, Q175FDN mice exhibit earlier onset and a greater variety and severity of HD-like phenotypes than Q175F mice or any previously reported KI HD mouse model, making them valuable for preclinical studies.

Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides.

Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD.

Repair of DNA double-strand breaks is not modulated by low-dose gamma radiation in C57BL/6J mice.

In this study, we sought to determine whether low-dose ionizing radiation, previously shown to induce a systemic adaptive response in C57BL/6J mice, is capable of enhancing the rate of DNA double-strand break repair. Repair capacity was determined by measuring γ-H2AX levels in splenic and thymic lymphocytes, using flow cytometry, at different times after a challenge irradiation (2 Gy, (60)Co). Irradiation with low doses (20 and 100 mGy) was conducted in vivo, whereas the challenge dose was applied to primary cultures of splenocytes and thymocytes in vitro 24 h later. Obtained kinetics curves of formation and loss of γ-H2AX indicated that cells from low-dose irradiated mice did not express more efficient DNA double-strand break repair compared to controls. Immunoblot analysis of γ-H2AX and Phospho-Ser-1981 ATM confirmed that DNA damage signaling was not modulated by preliminary low-dose radiation. Mouse embryonic fibroblasts of C57BL genetic background failed to show clonogenic survival radioadaptive response or enhanced repair of DNA double-strand breaks as evaluated by immunofluorescence microscopy of γ-H2AX foci. Our results indicate that radiation adaptive responses at systemic levels, such as increases in the tumor latency times in aging mice, may not be mediated by modulated DNA repair, and that the genetic background may affect expression of a radioadaptive response.

Activity-dependent secretion of progranulin from synapses.

The secreted growth factor progranulin (PGRN) has been shown to be important for regulating neuronal survival and outgrowth, as well as synapse formation and function. Mutations in the PGRN gene that result in PGRN haploinsufficiency have been identified as a major cause of frontotemporal dementia (FTD). Here we demonstrate that PGRN is colocalized with dense-core vesicle markers and is co-transported with brain-derived neurotrophic factor (BDNF) within axons and dendrites of cultured hippocampal neurons in both anterograde and retrograde directions. We also show that PGRN is secreted in an activity-dependent manner from synaptic and extrasynaptic sites, and that the temporal profiles of secretion are distinct in axons and dendrites. Neuronal activity is also shown to increase the recruitment of PGRN to synapses and to enhance the density of PGRN clusters along axons. Finally, treatment of neurons with recombinant PGRN is shown to increase synapse density, while decreasing the size of the presynaptic compartment and specifically the number of synaptic vesicles per synapse. Together, this indicates that activity-dependent secretion of PGRN can regulate synapse number and structure.