The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements
Abstract
:1. Introduction
2. Materials and Methods
2.1. Murine Erythroid Leukemia (MEL) HBBIVS Cells
2.2. Indel Characterization in Transgenic Humanized MEL-HBBIVS Cell Lines
2.2.1. Analysis of Indels in Bulk Cells
2.2.2. Analysis of Indels in Disrupted MEL-HBBIVS Clones
2.3. Sequencing
2.4. Assessment of RNA Expression
2.5. Globin Chain Analyses
2.6. Statistical and Bioinformatics Analyses
2.6.1. Statistical Analyses
2.6.2. Retrieval of Target Sequences
2.6.3. In Silico Design and Evaluation of Designer Nucleases
3. Results
3.1. Analysis of DARE in Bulk Populations of Modified Polyclonal MEL-HBBIVS Cells
3.2. Analysis of DARE in Cell Clones of Modified Clonal MEL-HBBIVS Cells
3.3. Analysis of Potential Alternative DARE Targets for β-Thalassemia
3.4. Additional Disease Targets for DARE
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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IthaID1 | Common Name | HGVS2 Name | Type of Mutation | Region | Exon⇥3 | References |
---|---|---|---|---|---|---|
107 | IVS I-5 G>A | HBB:c.92+5G>C | Activation of cSD4; partial SD5 LoF6 | SD-proximal | 5 nt7 | [31,32] |
111 | IVS I-6 T>C | HBB:c.92+6T>C | Activation of cSD; partial SD LoF | SD-proximal | 6 nt | [33] |
3445 | IVSI-13G | HBB:c.92+13 | cSD activated by IthaID 101-1038, 107, 111 | Intron | 13 nt | [31] |
113 | IVS I-110 G>A | HBB:c.93-21G>A | Confirmed target; GG>GA (aSA9) | aSA | 21 nt | [16,17] |
3008 | IVS I-115 A>T | HBB:c.93-16A>T | AT>TT (effect unclear) | Intron | 16 nt | [34] |
114 | IVS I-116 T>G | HBB:c.93-15T>G | TT>GT (potential aSD10) | aSD | 15 nt | [35] |
208 | IVS II-5 G>C | HBB:c.315+5G>C | Activation of cSD; partial LoF | Intron | 5 nt | [36,37] |
3446 | IVS II-579G | HBB:c.316-272 | cSA11 activated by IthaID 214 | Intron | 270 nt | [31] |
210 | IVS II-613 C>T | HBB:c.316-238C>T | Activation of cryptic splice site | Intron | 238 nt | [38] |
211 | IVS II-654 C>T | HBB:c.316-197C>T | Confirmed target; GC>GT (aSD) | Intron | 197 nt | [17,39] |
212 | IVS II-705 T>G | HBB:c.316-146T>G | Activation of cryptic splice site | Intron | 146 nt | [40,41] |
213 | IVS II-726 A>G | HBB:c.316-125A>G | Likely block of RNA processing | Intron | 125 nt | [42] |
214 | IVS II-745 C>G | HBB:c.316-106C>G | aSD, activating cSA IthaID 3446 | Intron | 106 nt | [31] |
215 | IVS II-761 A>G | HBB:c.316-90A>G | AT>GT (potential aSD) | Intron | 90 nt | ITHANET12 |
2183 | IVS II-781 C>G | HBB:c.316-70C>G | CT>GT (potential aSD) | Intron | 70 nt | [43] |
216 | IVS II-815 C>T | HBB:c.316-36C>T | CT>GT (potential aSD) | Intron | 36 nt | [44] |
217 | IVS II-837 T>G | HBB:c.316-14T>G | AT>AG (potential aSA) | Intron | 14 nt | [45] |
Primarily Affected | Exemplary | Exemplary Mutations | References | |||
---|---|---|---|---|---|---|
Organ System | Disorders | Gene | dbSNP ID | Effect1 | Frequency2 | |
Circulatory | Poikilocytic anemia | SPTA1 | rs757147440 | aSA | 25%3 | [47] |
Endocrine | Hyperinsulinemic hypoglycemia | ABCC8 | rs151344623 | aSA | 68.8%4 | [48,49] |
Nervous | Leber congenital amaurosis | CEP290 | rs281865192 | cSD activation | 43%5 | [50,51,52] |
Sensory | Stargardt disease | ABCA4 | rs1457937638 | cSD activation | 7.5%6 | [53,54] |
Muscular | Miyoshi myopathy | DYSF | rs1285082850 | cSD activation | 17 families7 | [55] |
Congenital muscular dystrophy | FKTN | rs1554754182 | cSD activation | 20.8%8 | [56] | |
Integumentary | Erythropoietic protoporphyria | FECH | rs2272783 | cSA activation | 42.6%9 | [57,58] |
Respiratory | Cystic fibrosis | CFTR | rs397508266 | aSD | 2.0%10 | [59,60] |
Multisystemic | Fabry disease | GLA | rs199473684 | cSD activation | 41.1%11 | [61,62] |
Cancer | Breast cancer | BRCA2 | rs191253965 | cSD activation | 0.2%12 | [63] |
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Patsali, P.; Mussolino, C.; Ladas, P.; Floga, A.; Kolnagou, A.; Christou, S.; Sitarou, M.; Antoniou, M.N.; Cathomen, T.; Lederer, C.W.; et al. The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements. J. Clin. Med. 2019, 8, 1959. https://doi.org/10.3390/jcm8111959
Patsali P, Mussolino C, Ladas P, Floga A, Kolnagou A, Christou S, Sitarou M, Antoniou MN, Cathomen T, Lederer CW, et al. The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements. Journal of Clinical Medicine. 2019; 8(11):1959. https://doi.org/10.3390/jcm8111959
Chicago/Turabian StylePatsali, Petros, Claudio Mussolino, Petros Ladas, Argyro Floga, Annita Kolnagou, Soteroula Christou, Maria Sitarou, Michael N. Antoniou, Toni Cathomen, Carsten Werner Lederer, and et al. 2019. "The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements" Journal of Clinical Medicine 8, no. 11: 1959. https://doi.org/10.3390/jcm8111959