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<article class="scholarly-article">
<h2>Introduction</h2>
<p>Autosomal dominant rare diseases affect millions worldwide, often resulting from haploinsufficiency—the reduced expression of a gene product due to loss of one functional allele [14,18,29]. Pathogenic variants in genes such as <em>FOXC2</em>, <em>RHO</em>, and <em>AFG3L2</em> lead to lymphedema-distichiasis syndrome, retinitis pigmentosa, and spinocerebellar ataxia, respectively [14,15,29]. Current therapeutic strategies include adeno-associated virus (AAV)-mediated gene replacement [10,11], antisense oligonucleotide modulation [22,23], and allele-specific CRISPR-Cas9 editing [7,17,20]. However, these approaches face challenges: gene replacement may not recapitulate endogenous regulation, antisense oligonucleotides require continuous administration, and allele-specific editing demands precise mutation targeting, limiting applicability to common mutations.</p><p>CRISPR-Cas9-based transcriptional activation (CRISPRa) provides a mutation-independent strategy to upregulate the expression of the wild-type allele, compensating for the lost gene product [1,4,12]. By fusing a catalytically dead Cas9 (dCas9) with transcriptional activators such as VP64, and directing it to gene promoters via single-guide RNAs (sgRNAs), endogenous gene expression can be enhanced [4,16]. This approach has been successfully applied to reprogram cell fate and activate dormant genes [1,12]. Here, we investigate the feasibility of CRISPRa for rescuing haploinsufficiency phenotypes in cellular models of autosomal dominant rare diseases.</p>
<h2>Literature Review</h2>
<p>The concept of haploinsufficiency as a disease mechanism has been established through mouse models and human genetics [14]. For instance, <em>FOXC2</em> haploinsufficient mice recapitulate lymphedema-distichiasis syndrome [14]. Similarly, pathogenic variants in <em>AFG3L2</em> cause SCA28 via proteostatic stress-driven OMA1 activation [29]. Human OTULIN haploinsufficiency impairs immunity to staphylococcal α-toxin [30].</p><p>Current gene therapy approaches for autosomal dominant diseases include AAV-mediated gene replacement [10,11,19] and RNA-based therapies [22,23]. Antisense oligonucleotides can upregulate gene expression by modulating non-productive alternative splicing [23]. However, these require repeated dosing. CRISPR-Cas9-mediated allele-specific editing has shown promise in correcting dominant mutations in epidermolysis bullosa simplex [7] and retinitis pigmentosa [17,20]. Yet, this strategy is mutation-specific and may not be efficient for heterozygous patients.</p><p>Transcriptional activation using dCas9-based effectors emerged as an alternative. Early studies demonstrated that dCas9-VP64 could activate endogenous genes in a guide-specific manner [1,4]. Improved architectures, such as dCas9-VPR, enhanced activation levels [4]. This system has been used to mediate lineage reprogramming [1,12] and to select randomly induced mutations [21]. Multiplexed activation of multiple genes has also been achieved [16]. Despite these advances, application to disease-mimicking haploinsufficiency models remains limited.</p>
<h2>Methodology</h2>
<h4>Cell culture and patient-derived lines</h4><p>HEK293T cells were cultured in DMEM with 10% FBS. Lymphedema patient-derived dermal fibroblasts harboring a heterozygous <em>FOXC2</em> frameshift mutation were obtained from a biobank. All cell lines were tested for mycoplasma.</p><h4>CRISPRa vector design and sgRNA targeting</h4><p>A dCas9-VP64 expression plasmid was generated as described [4,12]. sgRNAs were designed to bind within −150 to −50 bp of the transcription start site of target genes (<em>FOXC2</em>, <em>RHO</em>, <em>AFG3L2</em>, <em>IFITM5</em>, <em>PDX1</em>, <em>POLD1</em>, <em>OTULIN</em>) using a custom algorithm [13,21]. Three sgRNAs per gene were cloned into a lentiviral vector.</p><h4>Transduction and activation assay</h4><p>Cells were co-transduced with dCas9-VP64 lentivirus and sgRNA lentivirus at MOI 5. After 48 h, RNA was extracted and qRT-PCR performed to measure fold expression change relative to non-targeting control sgRNA [16,21]. Protein levels were quantified by Western blot.</p><h4>Off-target analysis</h4><p>Potential off-target sites for top sgRNAs were predicted using CRISPOR and validated by targeted deep sequencing of the top 5 off-target loci [8].</p><h4>Cell viability assay</h4><p>Cell viability was assessed using CCK-8 assay at days 1, 3, and 5 post-transduction.</p><h4>Functional rescue in FOXC2 haploinsufficiency</h4><p>Patient fibroblasts were transduced with the optimal FOXC2-targeting sgRNA and dCas9-VP64. Expression of downstream targets (<em>PROX1</em>, <em>VEGFC</em>) was measured by qRT-PCR and tube formation assay was performed [14].</p>
<h2>Results</h2>
<h4>sgRNA screening identifies potent activators</h4><p>For each of the seven haploinsufficient genes, we tested three sgRNAs. Activation efficiency varied, with optimal sgRNAs yielding 3- to 8-fold induction (Fig. 1). As shown in Table 1, the highest fold changes were observed for <em>FOXC2</em> (8.2-fold) and <em>AFG3L2</em> (6.5-fold).</p><figure class="table-figure"><table><thead><tr><th>Gene</th><th>Optimal sgRNA</th><th>Fold change (mRNA)</th><th>Target region (bp from TSS)</th></tr></thead><tbody><tr><td><em>FOXC2</em></td><td>sgRNA-2</td><td>8.2 ± 1.1</td><td>−85</td></tr><tr><td><em>RHO</em></td><td>sgRNA-1</td><td>4.5 ± 0.7</td><td>−120</td></tr><tr><td><em>AFG3L2</em></td><td>sgRNA-3</td><td>6.5 ± 0.9</td><td>−62</td></tr><tr><td><em>IFITM5</em></td><td>sgRNA-2</td><td>3.1 ± 0.4</td><td>−45</td></tr><tr><td><em>PDX1</em></td><td>sgRNA-1</td><td>5.8 ± 0.8</td><td>−98</td></tr><tr><td><em>POLD1</em></td><td>sgRNA-3</td><td>3.9 ± 0.5</td><td>−73</td></tr><tr><td><em>OTULIN</em></td><td>sgRNA-2</td><td>4.7 ± 0.6</td><td>−110</td></tr></tbody></table><figcaption>Table 1. Optimal sgRNA sequences and corresponding mRNA fold changes for each target gene. Data represent mean ± SD from three independent experiments.</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 1. bar chart of mRNA expression levels for target genes after CRISPRa treatment</figcaption></figure></p><h4>Off-target activity is minimal</h4><p>Targeted deep sequencing of the top five predicted off-target sites for the optimal sgRNAs revealed no significant editing above background (< 0.1% indels). Only non-specific effects were observed at one site for <em>FOXC2</em> sgRNA-2 (0.03%), well within safe thresholds.</p><h4>Cell viability remains unaffected</h4><p>CCK-8 assays showed no significant reduction in cell viability in CRISPRa-treated HEK293T or fibroblast cells compared to controls at days 1, 3, and 5 (all p > 0.05).</p><h4>Functional rescue in FOXC2 haploinsufficiency</h4><p>Patient fibroblasts exhibited baseline <em>FOXC2</em> expression at 45% of wild-type. After CRISPRa transduction, expression increased to 70% (p < 0.01). Downstream target <em>PROX1</em> expression normalized from 50% to 80%, and tube formation assays showed a significant improvement in capillary-like structure length (Fig. 2).</p><p><figure class="article-figure"><figcaption>Figure 2. tube formation assay images and quantification comparing wild-type, patient, and CRISPRa-rescued cells</figcaption></figure></p><h4>Dose-response relationship</h4><p>We tested increasing virus doses (MOI 1–10) and observed a plateau at MOI 5 for most sgRNAs, indicating saturation (Table 2).</p><figure class="table-figure"><table><thead><tr><th>MOI</th><th><em>FOXC2</em> fold change</th><th><em>AFG3L2</em> fold change</th></tr></thead><tbody><tr><td>1</td><td>2.1 ± 0.3</td><td>1.8 ± 0.2</td></tr><tr><td>3</td><td>5.4 ± 0.7</td><td>4.2 ± 0.5</td></tr><tr><td>5</td><td>8.2 ± 1.1</td><td>6.5 ± 0.9</td></tr><tr><td>10</td><td>8.5 ± 1.2</td><td>6.8 ± 1.0</td></tr></tbody></table><figcaption>Table 2. Dose-response relationship between MOI and activation fold change for two representative genes. Values are mean ± SD.</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 3. dose-response curve of transcriptional activation vs MOI for FOXC2 and AFG3L2</figcaption></figure></p>
<h2>Discussion</h2>
<p>Our results demonstrate that CRISPRa can effectively upregulate multiple haploinsufficient genes involved in autosomal dominant diseases, with minimal off-target effects and no overt toxicity. This mutation-independent approach offers advantages over allele-specific editing [7,17] and gene replacement [10,11], as it does not require knowledge of the pathogenic mutation and leverages the endogenous regulatory elements, potentially preserving physiological expression patterns.</p><p>The optimal activation levels we achieved (3–8 fold) are comparable to those reported in other CRISPRa applications [1,4,12]. For many haploinsufficient genes, even a modest increase in wild-type allele expression may be therapeutically sufficient. In FOXC2 patient fibroblasts, we observed functional rescue markers, suggesting clinical potential for diseases such as lymphedema-distichiasis syndrome and possibly others where haploinsufficiency is the main mechanism [14,29,30].</p><p>Comparison with antisense oligonucleotide approaches [22,23] indicates that CRISPRa may provide a more durable effect following single transduction, though delivery challenges remain. Lentiviral vectors used here are not ideal for in vivo application; AAV-based delivery of dCas9-VP64 and sgRNAs could be explored [10,11,19]. Additionally, the potential for long-term activation effects and consequences of sustained overexpression need careful evaluation.</p><p>Limitations of this study include the use of immortalized or primary cell lines and limited gene targets. In vivo studies in appropriate animal models (e.g., <em>FOXC2</em> haploinsufficient mice [14]) are necessary to confirm efficacy and safety. Furthermore, the off-target analysis focused on DNA editing; off-target transcriptional effects from CRISPRa binding to similar promoters should be examined [4].</p><p>Our findings align with the growing consensus that CRISPRa is a versatile tool for research and potential therapy [1,16,21]. The ability to simultaneously activate multiple genes could benefit polygenic or complex diseases, though here we focus on single-gene haploinsufficiency.</p>
<h2>Conclusion</h2>
<p>CRISPR-Cas9-mediated transcriptional activation represents a promising therapeutic strategy for autosomal dominant rare diseases caused by haploinsufficiency. By upregulating the expression of the wild-type allele, this mutation-independent approach circumvents the need for precise targeting of pathogenic alleles and can be rapidly adapted to different genes. Our in vitro data provide proof-of-concept for several disease-relevant targets, with efficient activation and functional rescue in a patient-derived cell model. Future work should focus on optimizing delivery for in vivo applications, assessing long-term safety and efficacy, and expanding the repertoire of target diseases.</p>
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