Robust, Durable Gene Activation In Vivo via mRNA-Encoded Activators

ACS nano

2022 Mar 31

Beyersdorf, JP;Bawage, S;Iglesias, N;Peck, HE;Hobbs, RA;Wroe, JA;Zurla, C;Gersbach, CA;Santangelo, PJ;
PMID: 35357116 | DOI: 10.1021/acsnano.1c10631

Programmable control of gene expression via nuclease-null Cas9 fusion proteins has enabled the engineering of cellular behaviors. Here, both transcriptional and epigenetic gene activation via synthetic mRNA and lipid nanoparticle delivery was demonstrated in vivo. These highly efficient delivery strategies resulted in high levels of activation in multiple tissues. Finally, we demonstrate durable gene activation in vivo via transient delivery of a single dose of a gene activator that combines VP64, p65, and HSF1 with a SWI/SNF chromatin remodeling complex component SS18, representing an important step toward gene-activation-based therapeutics. This induced sustained gene activation could be inhibited via mRNA-encoded AcrIIA4, further improving the safety profile of this approach.

The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy

Human gene therapy

2022 May 04

Wasala, NB;Million, ED;Watkins, TB;Wasala, LP;Han, J;Yue, Y;Lu, B;Chen, SJ;Hakim, CH;Duan, D;
PMID: 35350865 | DOI: 10.1089/hum.2021.130

Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Nat Med. 2019 Jan 21.

2019 Jan 21

Maeder ML, Stefanidakis M, Wilson CJ, Baral R, Barrera LA, Bounoutas GS, Bumcrot D, Chao H, Ciulla DM, DaSilva JA, Dass A, Dhanapal V, Fennell TJ, Friedland AE, Giannoukos G, Gloskowski SW, Glucksmann A, Gotta GM, Jayaram H, Haskett SJ, Hopkins B, Horng JE, Joshi S, Marco E, Mepani R, Reyon D, Ta T, Tabbaa DG, Samuelsson SJ, Shen S, Skor MN, Stetkiewicz P, Wang T, Yudkoff C, Myer VE, Albright CF, Jiang H.
PMID: PMID: 30664785 | DOI: DOI:10.1038/s41591-018-0327-9

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9.

J Control Release.

2019 Feb 13

Stephens CJ, Lauron EJ, Kashentseva E, Lu ZH, Yokoyama WM, Curiel DT.
PMID: 30771412 | DOI: 10.1016/j.jconrel.2019.02.009

Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein function and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene therapy approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in vector dilution and subsequent loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. Integration allows replication of the therapeutic transgene at every cell division and can avoid issues associated with vector dilution. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene knock-in. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of FIX activity and phenotypic correction in a murine model of juvenile HB. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. Overall, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease, suggesting this approach may be applicable to other diseases.

Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

Neuron

2019 Feb 18

Bäck S, Necarsulmer J, Whitaker LR, Coke LM, Koivula P, Heathward EJ, Fortuno LV, Zhang Y, Yeh CG, Baldwin HA, Spencer MD, Mejias-Aponte CA, Pickel J, Hoffman AF, Spivak CE, Lupica CR, Underhill SM, Amara SG, Domanskyi A, Anttila JE, Airavaara M, Hope BT, Hamra FK, Richie CT, Harvey BK.
PMID: 30792150 | DOI: 10.1016/j.neuron.2019.01.035

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation

Development (Cambridge, England)

2023 Mar 27

Elliott, KH;Balchand, SK;Bonatto Paese, CL;Chang, CF;Yang, Y;Brown, KM;Rasicci, DT;He, H;Thorner, K;Chaturvedi, P;Murray, SA;Chen, J;Porollo, A;Peterson, KA;Brugmann, SA;
PMID: 36971348 | DOI: 10.1242/dev.201237

Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. While the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity (https://research.cchmc.org/Ciliome_Gene_Expression/). Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by utilizing Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore long-standing questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.

Recent advancements in CRISPR-Cas toolbox for imaging applications

Critical reviews in biotechnology

2021 Aug 18

Singh, V;Jain, M;
PMID: 34407706 | DOI: 10.1080/07388551.2021.1950608

The imaging of chromatin, genomic loci, RNAs, and proteins is very important to study their localization, interaction, and coordinated regulation. Recently, several clustered regularly interspaced short palindromic repeats (CRISPR) based imaging methods have been established. The refurbished tool kits utilizing deactivated Cas9 (dCas9) and dCas13 have been established to develop applications of CRISPR-Cas technology beyond genome editing. Here, we review recent advancements in CRISPR-based methods that enable efficient imaging and visualization of chromatin, genomic loci, RNAs, and proteins. RNA aptamers, Pumilio, SuperNova tagging system, molecular beacons, halotag, bimolecular fluorescence complementation, RNA-guided endonuclease in situ labeling, and oligonucleotide-based imaging methods utilizing fluorescent proteins, organic dyes, or quantum dots have been developed to achieve improved fluorescence and signal-to-noise ratio for the imaging of chromatin or genomic loci. RNA-guided RNA targeting CRISPR systems (CRISPR/dCas13) and gene knock-in strategies based on CRISPR/Cas9 mediated site-specific cleavage and DNA repair mechanisms have been employed for efficient RNA and protein imaging, respectively. A few CRISPR-Cas-based methods to investigate the coordinated regulation of DNA-protein, DNA-RNA, or RNA-protein interactions for understanding chromatin dynamics, transcription, and protein function are also available. Overall, the CRISPR-based methods offer a significant improvement in elucidating chromatin organization and dynamics, RNA visualization, and protein imaging. The current and future advancements in CRISPR-based imaging techniques can revolutionize genome biology research for various applications.

Neurotensin orchestrates valence assignment in the amygdala

Nature

2022 Jul 20

Li, H;Namburi, P;Olson, JM;Borio, M;Lemieux, ME;Beyeler, A;Calhoon, GG;Hitora-Imamura, N;Coley, AA;Libster, A;Bal, A;Jin, X;Wang, H;Jia, C;Choudhury, SR;Shi, X;Felix-Ortiz, AC;de la Fuente, V;Barth, VP;King, HO;Izadmehr, EM;Revanna, JS;Batra, K;Fischer, KB;Keyes, LR;Padilla-Coreano, N;Siciliano, CA;McCullough, KM;Wichmann, R;Ressler, KJ;Fiete, IR;Zhang, F;Li, Y;Tye, KM;
PMID: 35859170 | DOI: 10.1038/s41586-022-04964-y

The ability to associate temporally segregated information and assign positive or negative valence to environmental cues is paramount for survival. Studies have shown that different projections from the basolateral amygdala (BLA) are potentiated following reward or punishment learning1-7. However, we do not yet understand how valence-specific information is routed to the BLA neurons with the appropriate downstream projections, nor do we understand how to reconcile the sub-second timescales of synaptic plasticity8-11 with the longer timescales separating the predictive cues from their outcomes. Here we demonstrate that neurotensin (NT)-expressing neurons in the paraventricular nucleus of the thalamus (PVT) projecting to the BLA (PVT-BLA:NT) mediate valence assignment by exerting NT concentration-dependent modulation in BLA during associative learning. We found that optogenetic activation of the PVT-BLA:NT projection promotes reward learning, whereas PVT-BLA projection-specific knockout of the NT gene (Nts) augments punishment learning. Using genetically encoded calcium and NT sensors, we further revealed that both calcium dynamics within the PVT-BLA:NT projection and NT concentrations in the BLA are enhanced after reward learning and reduced after punishment learning. Finally, we showed that CRISPR-mediated knockout of the Nts gene in the PVT-BLA pathway blunts BLA neural dynamics and attenuates the preference for active behavioural strategies to reward and punishment predictive cues. In sum, we have identified NT as a neuropeptide that signals valence in the BLA, and showed that NT is a critical neuromodulator that orchestrates positive and negative valence assignment in amygdala neurons by extending valence-specific plasticity to behaviourally relevant timescales.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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