1 Title: Novel Approaches for HTLV-1 Therapy: Innovative Applications of CRISPR-

2 Cas9
3

4 Abstract

5 The Human T-cell lLymphotropic vVirus type 1 (HTLV-1) is a single-stranded

6 positive-sense RNA virus that belongings to the deltaretrovirus family, which and
7 infects approximately 5 to 10 million people worldwide. While a significant number of
8 individuals living with HTLV-1 remain asymptomatic throughout their lives, some
9 develop one or more severe clinical conditions, such as tropical spastic paraparesis
10 (HAM/TSP), a progressive and debilitating disease, and/or a subtype of non-Hodgkin`s
11 lymphoma with a more ominousthreatening course known as adult T-cell
12 leukemia/lymphoma (ATLL). Moreover, current therapeutic options are limited, and
13 focus primarily focusing on treating symptoms management and controlling viral
14 latency. CRISPR-Cas9 gene editing is proposed as a promising tool to address the
15 intricate complexities links associated with HTLV-1. By targeting the knockout or
16 silencing of key genes during the initial infection process and the dysregulatingon of
17 immune system signaling pathways, CRISPR-Cas9 offers potential intervention
18 opportunitiesavenues for intervention. In this review, we delve addressinto the
19 therapeutic potential of CRISPR-Cas9 gene editing, examine ing tthe primary
20 mechanisms involved in editing potential target genes, and discussing the existing
21 evidence findings in the current ontemporary scientific literature.

22
23 Keywords: HTLV-1; CRISPR; CRISPR-Cas9; TAX; HBZ
24

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25 Introduction

26 The human T-lymphotropic virus type 1 (HTLV-1), a positive-sense single-

27 stranded RNA retrovirus belonging to the deltaretrovirus family, infects approximately
28 5 to 10 million people worldwide1. HTLV-1 mainlyPredominantly transmitted through
29 sexual contact, blood transfusion, and from mother to child via breastfeeding, HTLV-1
30 exhibits higher prevalence rates in specific regions like Japan, the Caribbean, and sub-
31 Saharan Africa2,3. While the majority of HTLV-1 infections remain asymptomatic, a
32 small percentage of carriers (around 3 to 5%) may develop debilitating clinical
33 conditions with uncertain prognosies, including tropical spastic paraparesis
34 (HAM/TSP), and/or adult T-cell leukemia/lymphoma (ATLL)4,5.

35 HAM/TSP is characterized by progressive neurological symptoms, such as

36 weakness and stiffness in the legs, urinary dysfunction, and sensory abnormalities 4,6.
37 These symptoms typically arise during the fourth or fifth decade of life, with a higher
38 incidence in women7. The pathogenesis of HAM/TSP involves a chronic inflammatory
39 response in the central nervous system, predominantly affecting the thoracic spinal cord.
40 Although the exact mechanisms underlying disease progression are not fully
41 understood, it is believed that the development of the disease can be categorized into
42 three main courses: rapid, mild, and slowly progressing, which involve both viral and
43 host factors6,8.

44 Furthermore, it was suggested by Haziot et al. 9, 2019, that HTLV-1-infected

45 patients, initially classified as asymptomatic, exhibited previously unrelated
46 neurological symptoms. These patients were then reclassified as carriers of an
47 intermediate syndrome (IS), indicating an intermediary clinical form between the
48 asymptomatic phase and HAM/TSP. Symptoms associated with IS included
49 neurological changes, visual disturbances, oral conditions, skin lesions, bladder
50 disturbances, and rheumatological symptoms. Additionally, the HTLV-1 proviral load,
51 bladder disturbances, and rheumatological symptoms were identified as independently
52 associated factors with IS9.

53 On the other hand, ATLL represents a malignancy originating from mature T-

54 cells. This condition is closely associated with profound immunosuppression, fostering
55 aggressive tumor growth and systemic manifestations such as fever, weight loss, and
56 lymphadenopathy. Despite intensive chemotherapy regimens, the prognosis for ATLL

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57 remains dismal, with the majority of patients succumbing to the disease within a short
58 time frame after diagnosis, between 5.5 and 13 months10. Furthermore, ATLL can be
59 classified into four subtypes according to Shimoyama criteria, wherein the acute and
60 lymphoma subtypes are notably aggressive, while chronic and smoldering ATLL tend
61 to exhibit a more indolent disease course11.

62 Numerous research endeavors are underway with the aim of identifying a

63 molecular entity crucial for determining the disease's unfavorable prognosis. Despite
64 remarkable progress, significant questions remain regarding the impacted immune
65 signaling pathways, as well as the insufficiency of genuinely efficacious therapeutic
66 interventions12–14. One of the most prominent strategies involves the utilization of
67 corticosteroids. However, while corticosteroids have shown effectiveness in alleviating
68 symptoms and controlling viral latency, their overall impact on the disease's progression
69 and patient outcomes remains uncertain and subject to debate15.

70 Concurrently, these studies are elucidating the intricate molecular mechanisms

71 behind diseases linked to HTLV-1, especially ATLL. These investigations have
72 revealed the dysregulation of various signaling pathways, spanning from T-cell
73 activation to survival, while also highlighting the pivotal role of viral factors in
74 modulating host immune responses and promoting oncogenesis16–18. Advancements in
75 understanding the genetic and epigenetic alterations associated with HTLV-1 infection
76 and ATLL development are fostering hope that precision medicine approaches can be
77 developed, tailored to individual patients' molecular profiles19,20.

78 The research findings suggest that the Tax protein plays a pivotal role in
79 dampening the transcriptional activation of IFN-β, a key antiviral cytokine, triggered by
80 the overexpression of STING (Stimulator of Interferon Genes). Tax exerts a negative
81 regulatory effect on the stimulated production of interferon-beta (IFN-β), mediated by
82 various molecules such as poly(dA:dT), interferon-stimulatory DNA (ISD), or cyclic
83 GMP-AMP (cGAMP). These molecules are integral components in the activation of the
84 cGAS/STING pathway, crucial for sensing cytosolic DNA and initiating innate immune
85 responses. The cGAS/STING pathway plays a pivotal role in orchestrating the
86 elimination of pro-inflammatory DNA and triggering cellular autophagy, thereby
87 contributing, or in this case failing to contribute, to the host's defense against viral
88 infections16,19.

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 89 Due to the dampened activation of IFN-β and subsequent impairment of the
90 Beclin 1 autophagy complex, accumulated pro-inflammatory DNA perpetuates the
91 sustained activation of the inhibitor of κB kinase (IKK) complex. This persistent
92 signaling leads to continuous nuclear factor kappa B (NF-kB) activation, triggering
93 prolonged inflammatory processes. Ultimately, this cascade directly contributes to
94 conditions such as human T-lymphotropic virus type 1-associated myelopathy
95 (HAM/TSP) and promotes cellular senescence associated with adult T-cell
96 leukemia/lymphoma (ATLL)16,19,20. ([Figure 1]).

97

98 Figure 1. Activation and iInhibition of cGAS/STING sSignaling during HTLV-1

99 iInfection.
100 Source: Adapted from Peng-Hui Zeng and Wen-Jun Yin, 2020.
101

102 The proviral genome of HTLV-1 is complex and contains several elements that
103 play crucial roles in the pathogenesis of the infection. In addition to Tax, another critical
104 element is the HTLV-1 basic leucine zipper factor (HBZ), an antisense transcript that is
105 approximately 9 kb in size. The expression of HBZ is meticulously regulated by
106 promoters located at the 5' and 3' ends of the virus's long terminal repeats (LTRs). This
107 finely tuned regulation allows HBZ to be consistently expressed in leukemic cells and to
108 perform functions of critical importance in the pathogenesis of ATL14.

109

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110 Unlike the Tax protein, whose expression varies, HBZ is consistently expressed
111 in ATL-affected cells, making it a pivotal component of the virus life cycle. HBZ is
112 strongly linked to the proviral load and significantly contributes to the proliferation of
113 infected T cells21–23. In vitro and animal model investigations have revealed that HBZ
114 interacts with multiple host factors, including c-Jun, JunB, JunD, CREB2, and CREB,
115 exerting a negative impact on the transcription of both viral and cellular genes 24,25.
116 ([Figure 2]).

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118
119 Figure 2. Expression and activities of HBZ RNA and protein. Representative
120 schematice of the HTLV-1 genome and its interactions with host factors. The
121 Eessential genes include gag, pro, pol, env, TAX-1, and HBZ. From tThese genes
122 encode, subproteins that are of fundamental importance for viral assembly and the
123 replication cycle are encoded. In tThe gag gene, codes for structural proteins such
124 as the capsid (CA), the matrix (MA), and the nucleocapsid (NC) are encoded. TIn
125 the pro gene, codes for proteins that are required for processing of necessary for viral
126 polyprotein processing are encoded. TIn the pol gene encodes, essential enzymes for
127 viral replication, such as reverse transcriptase (RT) and integrase (IN), are
128 encoded. The In the env gene, viral viral envelope proteins (SU-surface and TM-
129 transmembrane) are encoded by the env gene. Additionally, hHighlighted
130 interactions also include the negative-sense transcription of HBZ and its
131 relationship to with transcription factors such as c-Jun, JunB, and CREB2, which
132 influence ing hhost transcription factors and the immune response.
133
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135 Delving into the intricate role of HBZ within HTLV-1 infection stands as a
136 pivotal endeavor for the advancement of novel therapeutic interventions. The

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137 manipulation of HBZ gene dynamics and its interplay with cellular elements presents a
138 promising avenue to disrupt the advancement of Adult T-cell Leukemia/Lymphoma
139 (ATL), countering the deleterious effects propagated by Tax, and vice versa 24,25.
140 Consequently, delving into the workings of these genes holds the potential to shed light
141 on the underlying mechanisms driving the pathogenesis of various ailments linked to
142 HTLV-1. Such an approach is poised to induce significant shifts in the proliferation and
143 viability of infected cells, thereby dampening the immunomodulatory repercussions and
144 ultimately alleviating the burden of associated diseases23,25.

145 CRISPR-Cas9 Systems

146 The CRISPR-Cas9 system, a revolutionary discovery in molecular biology,

147 represents an adaptive defense mechanism found in bacteria and archaea. Its primary
148 function is to protect organisms against invasions of exogenous nucleic acids, such as
149 phages or plasmids14. By utilizing previously acquired immune memory of invasive
150 genetic elements, the CRISPR-Cas9 system is capable of recognizing and destroying
151 specific DNA or RNA sequences, providing an effective barrier against viral infections
152 and horizontal gene transfer26,27.

153 Since its discovery in 2012, the CRISPR-Cas9 system has been widely
154 recognized as an exceptionally powerful genome editing tool. Its ability to selectively
155 manipulate DNA sequences in a wide variety of organisms, including bacteria 28,
156 plants26, animals29, and even humans30,31, represents a significant advancement in
157 biotechnology. By directing a Cas protein to a specific DNA sequence, the CRISPR-
158 Cas9 system can induce precise cuts in the genome, allowing for the insertion, deletion,
159 or modification of genes with unprecedented accuracy. ([Figure 3)].

160

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161 Figure 3. illustrates the mechanism of action mechanism of the CRISPR/Cas9
162 system. (Figure 3aa): Depicts the recognition of theshows the target region recognition
163 of target region and the cleavage of the the double-stranded DNA cleavage based on
164 the sgRNA. Figure 3b(b): includes shows the DNA repair processes known as
165 nonhomologous end-joining (NHEJ) and homology-directed repair (HDR), along as
166 well as with essential system components such as the single-guide RNA (sgRNA)
167 and the protospacer adjacent motif (PAM). NHEJ is a DNA repair process in
168 which that directly joins the broken ends of DNA are directly joined together, often
169 leading to mutations introducing mutations in the process. On the other handcontrast,
170 HDR uses a homologous DNA sequence as a template to repair DNA double-strand
171 DNA breaks with high fidelity.
172
173

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174 Compared to other genome editing technologies such as meganucleases, ZFNs, and
175 TALENs, CRISPR-Cas stands out not only for its efficiency and precision but also
176 for its relative ease of use and affordability 32. Its fundamental basis lies in the base
177 complementarity between a guide RNA sequence (gRNA) and the genomic target,
178 giving it exceptional versatility in genomic engineering. In addition to its
179 applications in genome editing, the CRISPR-Cas9 system has been explored in a
180 variety of other areas, including molecular diagnostics, gene therapy, pest control,
181 and even data storage26,28,29,31.

182 The native CRISPR-Cas9 systems are classified into two main classes,
183 encompassing six types of Cas effector proteins. CRISPR-Cas9, belonging to class II
184 and type II, is the most prevalent and widely used system in CRISPR-based genome
185 editing strategies. CRISPR-Cas9 typically recognizes a specific sequence called PAM
186 (Protospacer Adjacent Motif), commonly known as NGG (where N can be any
187 nucleotide), which is essential for its function. In addition to CRISPR-Cas9, other
188 notable Cas proteins include Cas12a, type V, which also recognizes specific PAM
189 sequences, and Cas13, type VI, capable of precisely targeting RNA33.

190 This specific recognition of the sequence by the CRISPR-Cas9 system, such as
191 PAM or NGG for spCas9, is mediated by 17-20 nucleotides at the 5' end of the guide
192 RNA (gRNA). This specificity prevents undesired off-target interactions. The nuclease
193 domains of Cas9, HNH and RuvC, cleave the target and non-target DNA strands,
194 respectively. In the absence of a template, the resulting double-strand break is repaired
195 through the NHEJ pathway, introducing random indel mutations. Alternatively, the
196 HDR pathway uses an exogenous DNA repair template to introduce precise mutations at
197 a specific locus34.
198

199
200 CRISPR-Cas9 and HTLV-1

201 When exploring the potential of the CRISPR-Cas9 system in the treatment of
202 viral infections, such as HTLV-1, it is crucial to identify strategic genetic 14,27,31. Genes
203 essential for viral replication, such as tax and hbz, and host-specific genes, can be
204 precisely selected. Suppressing these genes may lead to effective inhibition of viral
205 replication, representing a crucial step towards a functional cure. This precise approach

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206 in defining target sequences is vital for successfully guiding therapeutic strategies,
207 offering the prospect of significant advances in the treatment of HTLV-1 infection.

208 For example, a study conducted by Prawiro et al. in 2023 provided an illustrative
209 case by identifying a single copy of the hotspot mutation PLCγ1 S345F in an ATL-
210 derived cell lineage. Through CRISPR-Cas9-mediated homology-directed repair
211 (HDR), they rectified the single nucleotide gene mutation. Remarkably, this restoration
212 to the wild-type PLCγ1 configuration adversely affected crucial functional properties of
213 the ATL cell lineage, notably its heightened proliferation rate and chemotaxis. These
214 findings, combined with insights gained from inhibiting the PLCγ1 pathway in the ATL
215 cell lineage, underscore the critical role of mutated PLCγ1 activation in ATL
216 pathology35.

217 Recently, the vasodilator-stimulated phosphoprotein (VASP) has emerged as a

218 pivotal player in facilitating the efficient transfer of p8 and HTLV-1 Gag, as elucidated
219 by Donhauser et al. (2020). The interaction site was pinpointed within the Ena/VASP
220 homology domain 1 (EVH1) of VASP, highlighting its critical role in mediating these
221 viral processes. Utilizing CRISPR-Cas9 methodology, researchers successfully
222 suppressed VASP expression, resulting in a significant decrease in p8 transfer both to
223 the cell surface and to Jurkat T cells. Conversely, despite expectations, the removal of
224 the CCCTC-binding factor (CTCF) did not yield conspicuously visible effects on viral
225 transcription or the examined epigenetic modifications36.

226 Furthermore, studies by Mohanty et al. (2020) and Fochi et al. (2019) revealed
227 crucial insights into the molecular mechanisms underlying NF-κB activation and viral
228 protein regulation in HTLV-1 pathogenesis. Mohanty, elucidated the pivotal role of
229 UBE4B in NF-κB activation and HTLV-1 Tax polyubiquitination, emphasizing its
230 potential as a therapeutic target. They demonstrated UBE4B's influence on both K48
231 and K63-linked polyubiquitination, implicating its significance in Tax-induced NF-κB
232 pathway activation37. Complementarily, Fochi investigated TRAF3's interaction with
233 HTLV-1 regulatory proteins, unveiling its impact on Tax-mediated NF-κB activation.
234 Notably, they highlighted APH-2's superiority over HBZ in hindering Tax-dependent
235 NF-κB activation, suggesting distinct roles for viral regulators. Together, these findings
236 deepen our understanding of HTLV-1 pathogenesis, offering promising avenues for
237 therapeutic intervention38.

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238 In parallel, recent investigations into monoclonal antibody (mAb) development
239 have presented promising avenues for targeted therapies against HTLV-1. By
240 characterizing antibodies such as BF4, capable of staining viral biofilms in HTLV-1-
241 infected T cells, researchers have made significant strides in identifying potential targets
242 for mAb-based interventions. Utilizing gene-based methods like CRISPR-Cas9,
243 researchers have validated these antibodies with restricted reactivities, offering a novel
244 approach to mAb validation. The identification of BF4 as an anti-CD82 mAb through
245 the CRISPR-Cas9 genomic-scale library (GeCKO) demonstrates the potential of this
246 approach in identifying therapeutic targets for HTLV-1-associated conditions. By
247 complementing traditional methods based on proteins and genes, this strategy provides a
248 promising alternative for the validation of mAbs and represents a significant step
249 forward in the development of targeted therapies against HTLV-139.
250
251 HTLV-1 and STLV-1
252
253 The human T-cell lymphotropic viruses Type 1 and 2 (HTLV-1 and HTLV-2),
254 along with their simian counterparts (STLV-1 and STLV-2), are part of the primate T-
255 cell lymphotropic virus group (PTLV). The high genetic similarity between HTLV-1
256 and STLV-1 strains suggests that the majority of HTLV-1 subtypes originate from
257 interspecies transmissions between monkeys and humans 1,40. Natural infection with
258 STLV-1 leads to the expression of viral proteins such as Tax and SBZ, which are
259 homologous to human proteins HBZ and Tax, respectively, and influence viral signaling
260 pathways LTR and NF-κB41.

261 The application of the CRISPR technique in animal models closely related to
262 humans can be directed to selectively interrupt the expression of these viral proteins.
263 This could allow the investigation of the impact of inhibiting these proteins on the
264 progression of STLV-1 infection, providing valuable information on potential
265 therapeutic strategies in humans42. The genetic similarity between HTLV-1 and HTLV-
266 2 subtypes is approximately 60% in nucleic acids and 70% in amino acids, indicating
267 independent evolution from the transmission of Simian T-Cell Lymphotropic Virus
268 (STLV) from non-human primates to humans 43. The endemicity of HTLV-1 and HTLV-
269 2 in remote populations suggests human infections over thousands of years. In

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270 particular, the Efe Mbuti Pygmies (Congo), considered descendants of proto-African
271 peoples, show infection with both viruses44.

272 The separation between African and non-African populations, estimated 75,000-
273 287,000 years ago, suggests a predominant gene flow from Pygmies to neighboring
274 populations. These pieces of evidence indicate ancient infections or more recent
275 interspecies transmissions of STLVs among Pygmies. Notably, frequent transmission of
276 STLV-1 from monkeys to humans in Africa has been documented, and infection with
277 HTLV-2 in Pygmies suggests an ancient history, with no related STLV-2 identified.
278 These observations highlight the evolutionary and transmission differences between
279 STLV and HTLV viruses40.

280 The high genetic similarity between HTLV-1 and STLV-1 strains, indicative of
281 interspecies transmission between monkeys and humans, suggests the use of the
282 CRISPR approach in developing specific strategies for these viruses. With CRISPR-
283 Cas9 technology, precise editing of genetic sequences would be possible, allowing
284 modulation of viral gene expression, deactivation of key virus components, or
285 elimination of viral genetic material from infected cells. In the context of STLVs, the
286 CRISPR technique can be employed to understand genetic differences and develop
287 specific intervention strategies aimed at limiting or eliminating interspecies
288 transmission of these viruses to humans40,42,42.

289 Challenges in the Pprecise Utilization use of CRISPR

290 While the results achieved so far are impressive and the use of genome editing
291 (GE) in various living organisms, including important agricultural crops, has become
292 commonplace, it is imperative to consider the inherent challenges in the precise use of
293 the CRISPR-Cas9 system. Issues related to the fidelity of CRISPR-Cas9 systems are
294 constantly addressed, especially concerning off-target mutations 45,46. These concerns are
295 of utmost importance due to the potential unintended consequences, emphasizing the
296 need for precise and reliable genetic modifications.

297 The CRISPR-Cas9 system, although widely used in genome editing due to its
298 effectiveness, is more prone to off-target effects compared to dimeric systems like
299 TALENs and ZFNs, owing to the monomeric nature of Cas9. These undesired effects
300 occur when the Cas9 complex binds to unintended regions, triggering cleavage events

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301 and potentially resulting in unintended genetic alterations. Strategies are being
302 developed to enhance the specificity of gRNA sequences, aiming to identify and
303 mitigate these potentially problematic regions 46. To address these challenges, strategies
304 involving modifications in the Cas9 component, adjustments in gRNA, and the use of
305 base editors, such as ribonucleoprotein complexes (RNP), are being explored to ensure
306 more precise and reliable genetic modifications.

307 Off-target effects in CRISPR-Cas9 systems can be classified into three main
308 types. The first involves regions with other motifs adjacent to the protospacer adjacent
309 motif (PAMs) containing substitutions or mismatches 47,48. The second type encompasses
310 regions with PAMs containing insertions or deletions (indels) compared to the DNA or
311 gRNA-target spacer, leading to the formation of a small bulge 49. The third type refers to
312 the cleavage of sequences with different PAM sites. Additionally, two categories of off-
313 target effects by CRISPR are proposed, distinguishing between those expected in
314 genomic regions with high sequence similarity to the target and those unexpected in
315 unrelated genomic regions47,48.

316 An overview: Recent advances and challenges in CRISPR-Cas9-mediated genome

317 editing for HTLV-1
318 Recent studies cited here demonstrate the potential of CRISPR-Cas9-mediated
319 genome editing in precisely modifying genetic sequences associated with HTLV-1
320 pathogenesis. For instance, the correction of specific mutations in ATL-derived cell
321 lineages through homology-directed repair (HDR) has shown significant adverse effects
322 on cellular functional properties35–38. These findings, along with others reported and
323 discussed in the literature, underscore the promise of targeted gene therapy to halt the
324 pathological processes associated with HTLV-1 infection.

325 However, despite significant advancements, technical and ethical challenges

326 persist in utilizing CRISPR-Cas9 technology to treat HTLV-1 infection. The issue of
327 guide RNA (gRNA) sequence specificity and the minimization of off-target effects are
328 areas of intensive focus. Researchers have been dedicated to enhancing CRISPR
329 specificity through the development of Cas9 enzyme variants with greater selectivity
330 toward the desired target46–49.

331 Furthermore, screening and validation techniques are essential to identify

332 potential off-targets, thus ensuring the accuracy of genetic editing 50. Another challenge

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333 lies in the efficient delivery of CRISPR technology to target cells, along with the
334 selection of appropriate cell lineages 51. Moreover, even after the identification of
335 suitable cell lineages, ensuring efficient delivery and expression of the CRISPR-Cas9
336 system in these cells is crucial, which can be complicated due to the heterogeneity of
337 cell cultures and the difficulty of transduction in primary or established cells 52. This
338 effective delivery is crucial for precisely targeting desired genetic modifications and
339 avoiding unwanted off-target effects, as mentioned earlier.

340 Genetic therapy requires the precise delivery of the CRISPR-Cas9 system to the
341 specific site where genetic editing is necessary. This process can be especially
342 challenging in hard-to-reach organs such as the brain or heart 53,54. Recent advances in
343 delivery technologies, such as viral vectors and nanoparticles, have shown promising
344 results; however, it is necessary to refine such methods to make them sufficiently safe
345 and effective for clinical application 55–57. Additionally, it is crucial to ensure that gene
346 editing in human cells is safe and does not induce long-term adverse effects. Clinical
347 trials must be carefully designed and conducted to assess the safety and efficacy of
348 CRISPR therapies before their widespread adoption. Furthermore, ethical
349 considerations, such as safety and equity in access to gene therapy, require continuous
350 attention to ensure the responsible application of CRISPR-Cas9 technology.

351 As research continues to advance, it is expected that CRISPR-Cas9 technology

352 will play an increasingly important role in the development of targeted therapies for
353 HTLV-1 infection. Innovative strategies, such as modulation of viral and host-specific
354 genes, have the potential to revolutionize the treatment of conditions associated with
355 HTLV-1, offering new hope for patients affected by this debilitating disease, who
356 currently lack effective therapies.

357

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