Brain transcriptome analysis of a CLN2 mouse model as a function of disease progression.

BACKGROUND: Neuronal ceroid lipofuscinoses, (NCLs or Batten disease) are a group of inherited, early onset, fatal neurodegenerative diseases associated with mutations in 13 genes. All forms of the disease are characterized by lysosomal accumulation of fluorescent storage material, as well as profound neurodegeneration, but the relationship of the various genes' function to a single biological process is not obvious. In this study, we used a well-characterized mouse model of classical late infantile NCL (cLINCL) in which the tripeptidyl peptidase 1 (Tpp1) gene is disrupted by gene targeting, resulting in loss of detectable TPP1 activity and leading to progressive neurological phenotypes including ataxia, increased motor deficiency, and early death. METHODS: In order to identify genes and pathways that may contribute to progression of the neurodegenerative process, we analyzed forebrain/midbrain and cerebellar transcriptional differences at 1, 2, 3 and 4 months of age in control and TPP1-deficient mice by global RNA-sequencing. RESULTS: Progressive neurodegenerative inflammatory responses involving microglia, astrocytes and endothelial cells were observed, accompanied by activation of leukocyte extravasation signals and upregulation of nitric oxide production and reactive oxygen species. Several astrocytic (i.e., Gfap, C4b, Osmr, Serpina3n) and microglial (i.e., Ctss, Itgb2, Itgax, Lyz2) genes were identified as strong markers for assessing disease progression as they showed increased levels of expression in vivo over time. Furthermore, transient increased expression of choroid plexus genes was observed at 2 months in the lateral and fourth ventricle, highlighting an early role for the choroid plexus and cerebrospinal fluid in the disease pathology. Based on these gene expression changes, we concluded that neuroinflammation starts, for the most part, after 2 months in the Tpp1-/- brain and that activation of microglia and astrocytes occur more rapidly in cerebellum than in the rest of the brain; confirming increased severity of inflammation in this region. CONCLUSIONS: These findings have led to a better understanding of cLINCL pathological onset and progression, which may aid in development of future therapeutic treatments for this disease.

Targeted genome editing for the correction or alleviation of primary Immunodeficiencies.

Primary immunodeficiencies (PID) are a growing list of unique disorders that result in a failure of the innate/adaptive immune systems to fully respond to disease or infection. PIDs are classified into five broad categories; B cell disorders, combined B and T cell disorders, phagocytic disorders, complement disorders, and disorders with recurrent fevers and inflammation. Many of these disorders, such as X-SCID, WAS, and CGD lead to early death in children if intervention is not implemented. At present, the predominant method of curative therapy remains an allogeneic transplant from a healthy donor, however many complications and limitations exist with his therapy such as availability of donors, graft vs host disease, graft rejection, and infection. More recently, gene therapy using viral based complementation vectors have successfully been implemented to functionally correct patient cells in an autologous transplant, but these methods carry significant risks, including insertional mutagenesis, and provide non-physiological gene expression. For these reasons, gene-editing reagents such as targeted nucleases, base editors (BE), and prime editors (PE) are being explored. The BE and PE tools, sometimes referred to as digital editors, are of very high interest as they provide both enhanced molecular specificity and do not rely on DNA repair pathways after DSBs to change individual base pairs or directly replace DNA sequences responsible for pathogenic phenotypes. With this in mind the purpose of this chapter is to highlight some of the most common PIDs found within the human population, discuss successes and shortcomings of previous intervention strategies, and highlight how the next generation of gene-editing tools may be deployed to directly repair the underlying genetic causes of this class of disease.

CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells.

CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR ( http://z.umn.edu/spliceR ) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.

Global Brain Transcriptome Analysis of a Tpp1 Neuronal Ceroid Lipofuscinoses Mouse Model.

In humans, homozygous mutations in the TPP1 gene results in loss of tripeptidyl peptidase 1 (TPP1) enzymatic activity, leading to late infantile neuronal ceroid lipofuscinoses disease. Using a mouse model that targets the Tpp1 gene and recapitulates the pathology and clinical features of the human disease, we analyzed end-stage (4 months) transcriptional changes associated with lack of TPP1 activity. Using RNA sequencing technology, Tpp1 expression changes in the forebrain/midbrain and cerebellum of 4-month-old homozygotes were compared with strain-related controls. Transcriptional changes were found in 510 and 1,550 gene transcripts in forebrain/midbrain and cerebellum, respectively, from Tpp1-deficient brain tissues when compared with age-matched controls. Analysis of the differentially expressed genes using the Ingenuity™ pathway software, revealed increased neuroinflammation activity in microglia and astrocytes that could lead to neuronal dysfunction, particularly in the cerebellum. We also observed upregulation in the production of nitric oxide and reactive oxygen species; activation of leukocyte extravasation signals and complement pathways; and downregulation of major transcription factors involved in control of circadian rhythm. Several of these expression changes were confirmed by independent quantitative polymerase chain reaction and histological analysis by mRNA in situ hybridization, which allowed for an in-depth anatomical analysis of the pathology and provided independent confirmation of at least two of the major networks affected in this model. The identification of differentially expressed genes has revealed new lines of investigation for this complex disorder that may lead to novel therapeutic targets.