Table 1.

Digital PCR (dPCR) Quantification of Δ5253-Corrected Dystrophin Genomes and Transcripts

DNA							Δ5253 (% Total Genomes)
Treatment		Vector Dose N/T/μD (×1013) (vg/vg/vg)		Treat. Age (Weeks)		Post-treat. (Weeks)	Heart		Diaphragm		Gastroc		TA		Liver
CRISPR		(a): 1/0.2/–		11		12	0.59 ± 0.07		0.043 ± 0.003		0.023 ± 0.007		–		–
		(b): 0.2/1/–		11		12	1.42 ± 0.06		0.066 ± 0.008		0.060 ± 0.020		–		–
		(c): 0.5/0.5/–		11		12	1.23 ± 0.21		0.082 ± 0.022		0.085 ± 0.009		–		–
		(d): 1/1/–		11		12	1.88 ± 0.16		0.137 ± 0.018		0.220 ± 0.021		–		0.000a
CRISPR		0.5/0.6/–		2		4	2.39 ± 0.51b		0.180 ± 0.060b		0.29 ± 0.150		0.153a		–
		0.5/0.6/–		2		18	2.31 ± 0.14		0.015 ± 0.004		0.072 ± 0.042		0.032a		–
CRISPR		0.5/1/–		2		18	2.57 ± 0.18		0.068 ± 0.016		0.077 ± 0.029		–		–
CRISPR + μDys		0.5/1/0.2		2		18	2.50 ± 0.11		0.623 ± 0.123		0.984 ± 0.101		–		–
Target + μDys		–/1/0.2		2		18	0.004 ± 0.004b		0.004 ± 0.004b		0.005 ± 0.005b		–		–
mRNA – Δ5253 Transcripts (% Total Dystrophin Transcripts)
Mouse #	Heart						Diaphragm				Gastroc
	CRISPR		CRISPR + μDys		Target + μDys		CRISPR	CRISPR + μDys		Target + μDys	CRISPR	CRISPR + μDys		Target + μDys
1	34.341		33.531		0.064		0.517	7.440		0.039	0.209	8.852		0.017
2	36.565		36.024		0.09		0.339	6.791		0.025	0.451	10.236		0.031
3	39.486		39.21		0.123		0.728	11.545		0.048	0.201	9.141		0.064
Mean	36.797		36.255		0.094		0.528	8.652		0.037	0.287	9.410		0.037
SEM	1.490		1.643		0.021		0.112	1.453		0.008	0.082	0.422		0.017

(Top) The percentage of genomes lacking exons 52-53 relative to total genomes for selected tissues of the different treatment groups, including heart, diaphragm, gastrocnemius (Gastroc), tibialis anterior (TA), and liver. Columns represent the following: nuclease (N), target (T), and μDys (μD) vector dose (×10¹³ vg); treatment age; time post-treatment; and percentage of genomes containing the Δ5253 deletion out of total genomes (encompassing genomes isolated from whole tissues, which include cells of myogenic, fibroblastic, adipocytic, and hematopoietic lineages, etc.). Vector dose values (vg/vg/vg) correspond to the delivered amount of nuclease, target, or μDys vectors (see Figure 1), respectively. Each value represents tissues obtained from three mice (n = 3) except where otherwise stated. (Bottom) Quantification of the percentage of dystrophin transcripts lacking exons 52-53 in heart, diaphragm, and gastrocnemius muscles of mice treated with nuclease and target vectors (CRISPR); nuclease, target, and microdystrophin vectors (CRISPR + μDys); or target and μDys vectors only (Target + μDys). RT-dPCR was performed on mRNA that was isolated from the same mice shown in the bottom three rows of the top (DNA) part of the table (n = 3). Values are represented as mean ± SEM. Dashes represent either that the μDys vector was not delivered or that samples were not analyzed. Decimal places are derived from each dPCR chip analysis, 25,000 genomes per sample, and most conditions were measured in triplicate.

^an = 1.

^bn = 2.