Review Reports - Block-and-Lock Approaches for HIV Cure: Mechanistic Insights, Challenges, and Emerging Role of CPSF6

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The submission provided "Block-and-Lock Approaches for HIV Cure: Mechanistic Insights, Challenges, and Emerging Role of CPSF6" provides a systematic review of mechanisms for "block and lock" approaches that affect integration-site targeting via LEDGF and CPSF6/LAD biology. This report is generally a thorough and valuable resource, but major revisions are needed to make it suitable for publication, including structure & formatting, presentation of the strength of available evidence, and claims made about clinical developments.

The review is a bit long and probably can be shortened for clarity throughout.
In the introduction, LEDGINs are introduced with recent human trials cited, but little provided as to WHICH such molecules are currently in trials. Some amount of discussion should be placed on Pirmitegravir at least. The information on BI-224436 is pretty dated and not the current state of the art.
It should also be acknowledged and discussed that the primary effect of LEDGINs is in LATE replication by a unique and well established mechanism, hence not the ideal mechanism to affect integration site selection.
More scientific caution should be exercised in citing clinical outcomes reported from conference-level preliminary results and the literature, for example the CRISPR subsection citing results for EBT-101.
In the Post-transcriptional and gene-silencing section, some comments should be made on current translation barriers for splicing inhibitors and RNAi, including feasibility of delivery, safety, and measurement.
In the section covering cellular transcription factor modulators, considering discussing why transcription-factor modulation is inherently less durable than integration site targeting, on a mechanistic and evidence based level.
the Kinase inhibitor section reads like a catalog rather than employing a narrative style. This should be revised extensively.
The LAD/CPSF6 section is the strongest aspect of this review. However, it is important to moderate on rhetoric such as “unique and unprecedented opportunity,” and “captivating possibility”. Bound these claims in this section and more broadly across the review, including the conclusions.
With regards to tone and claims in the Conclusions, place more emphasis on knowns vs unknowns and key uncertainties such as doses needed for integration retargeting, detability in the reservoir, and other potential confounding matters that need t be addressed.

Comments on the Quality of English Language

There are formatting and grammatical errors throughout the submission that must be addressed.

Author Response

Responses to Reviewer 1

Comments 1: The review is a bit long and probably can be shortened for clarity throughout.

Response 1: We acknowledge that the manuscript is relatively extensive, which reflects the complexity and breadth of the “block-and-lock” strategy as a therapeutic approach to HIV infection. In particular, the topic encompasses multiple mechanistic and therapeutic aspects that require comprehensive discussion. Moreover, we introduced a novel perspective on the role of CPSF6, which we aimed to address thoroughly by covering its various potential implications within this context. Nevertheless, in the revised version of the manuscript we have made a concerted effort to improve clarity and readability by simplifying several sections. We have substantially reduced the part concerning the kinase inhibitors and partially shortened the Conclusions section.

Comments 2: In the introduction, LEDGINs are introduced with recent human trials cited, but little provided as to which such molecules are currently in trials. Some amount of discussion should be placed on Pirmitegravir at least. The information on BI-224436 is pretty dated and not the current state of the art.

Response 2: We have considered this Reviewer’s point. In the Introduction, our intention was to provide only a concise overview of LEDGINs; however, in the revised version we have now explicitly included the names of the two compounds that have entered clinical evaluation. A more detailed discussion is provided in the dedicated section on LEDGINs. In particular, we have substantially expanded the section to include a more comprehensive description of pirmitegravir, which represents the most advanced compound currently under clinical investigation. In contrast, BI-224436 is discussed more briefly, as its clinical development has been discontinued despite previously promising results. We hope that this clarification and the revised manuscript adequately address the Reviewer’s concern.

Comments 3: It should also be acknowledged and discussed that the primary effect of LEDGINs is in LATE replication by a unique and well established mechanism, hence not the ideal mechanism to affect integration site selection.

Response 3: We agree with the Reviewer that the primary and best-established effect of LEDGINs occurs during the late phase of the HIV-1 replication cycle, where they impair proper virion maturation through a well-characterized mechanism, and we acknowledge that this represents their predominant antiviral activity. In response to this comment, we have revised the manuscript to explicitly include this important aspect within the section dedicated to LEDGINs. Specifically, we have incorporated a discussion of their late-stage mechanism of action and its biological implications.

Comments 4: More scientific caution should be exercised in citing clinical outcomes reported from conference-level preliminary results and the literature, for example the CRISPR subsection citing results for EBT-101.

Response 4: Following the Reviewer’s suggestion, we have revised the manuscript to clarify that the clinical data for EBT‑101 cited in the reference are preliminary and early-stage. Specifically, we now describe these findings as “preliminary early clinical data indicate…” and have added a statement highlighting that further clinical studies are required to confirm these observations.

Comments 5: In the post-transcriptional and gene-silencing section, some comments should be made on current translation barriers for splicing inhibitors and RNAi, including feasibility of delivery, safety, and measurement.

Response 5: In the revised version of the manuscript, we have incorporated in the section 3.1. and 3.2. a discussion of the current translational barriers for splicing inhibitors and RNAi within the Post-transcriptional and Gene-silencing section. Specifically, we now address challenges related to delivery, safety, and measurement, providing a more comprehensive overview of the practical considerations for these approaches. We believe that these additions enhance the clarity and completeness of the discussion in this section.

Comments 6: In the section covering cellular transcription factor modulators, considering discussing why transcription-factor modulation is inherently less durable than integration site targeting, on a mechanistic and evidence based level.

Response 6: In response to the Reviewer’s comment, we have revised the section to explicitly discuss the mechanistic differences between transcription-factor modulation (e.g., ZL0580) and integration site-targeting strategies. Specifically, we now highlight that transcription-factor modulators primarily affect proviral activity without altering its genomic location, which may limit the permanence of silencing compared to approaches that reposition proviruses into transcriptionally repressive chromatin. We also incorporated evidence that combination strategies, such as ZL0580 with LEDGINs, produce additive effects in blocking HIV-1 reactivation in both cell lines and primary cells, supporting the rationale for multi-target “block-and-lock” approaches.

Comments 7: the Kinase inhibitor section reads like a catalog rather than employing a narrative style. This should be revised extensively.

Response 7: We thank the Reviewer for this valuable suggestion. In the revised manuscript, we have extensively reduced and restructured the section on kinase inhibitors to move away from a catalog-style presentation. Rather than listing compounds in separate subsections, we now present the discussion in a continuous, narrative format that integrates mechanistic rationale, examples of representative inhibitors, and preclinical evidence.

Comments 8: The LAD/CPSF6 section is the strongest aspect of this review. However, it is important to moderate on rhetoric such as “unique and unprecedented opportunity,” and “captivating possibility”. Bound these claims in this section and more broadly across the review, including the conclusions.

Response 8: In the revised manuscript, we have moderated the language in the section discussing lenacapavir and LAD/CPSF6. Phrases such as “unique and unprecedented opportunity” and “captivating possibility” have been replaced with more neutral wording, while retaining the scientific content regarding the ability of lenacapavir to bias HIV-1 integration and the potential implications for promoting a transcriptionally repressed reservoir.

Comments 9: With regards to tone and claims in the Conclusions, place more emphasis on knowns vs unknowns and key uncertainties such as doses needed for integration retargeting, detability in the reservoir, and other potential confounding matters that need t be addressed.

Response 9: According to the Reviewer’s suggestion, we have moderated the tone of the Conclusions to emphasize the distinction between what is currently known and the key uncertainties that remain. We now highlight limitations and open questions regarding integration site modulation, including the doses required to achieve retargeting, the detectability of changes within the viral reservoir, and potential confounding factors that could influence outcomes. Statements regarding the translational potential of capsid inhibitors, such as lenacapavir, have been revised to adopt a more cautious, evidence-based tone, while retaining the discussion of mechanistic rationale and conceptual framework for block-and-lock strategies.

Comments 10: There are formatting and grammatical errors throughout the submission that must be addressed.

Response 10: We thank the Reviewer for highlighting this issue. In the revised manuscript, we have carefully reviewed the text and corrected all formatting and grammatical errors to ensure clarity and consistency throughout.

Reviewer 2 Report

Comments and Suggestions for Authors

Most efforts toward an HIV cure have been invested for strategies aimed at reversing viral latency to expose the infected cell to elimination via viral cytopathic effects or immune clearance (shock and kill). Genome editing via CRISPR represents an additional approach to an HIV cure that has been extensively investigated.

In their manuscript, Tolomeo and Cascio discuss current approaches to achieve an HIV cure via permanent silencing of the provirus, collectively known as block-and-lock. This is a major strength of the manuscript because the block-and-lock strategy remains underexplored, and this review shines new light on this alternative approaches. Additional strengths of the manuscript are the breadth and depth of the topics discussed. The authors cover a very broad range of potential host and viral factors that could be the target of block-and-lock strategies. They provide very detailed description of the functional role of each factor, potential approaches to target them along with a thoughtful analysis of their strengths and weaknesses. The authors focus in particular on strategies targeting the host factors LEDGF/p75 and CPSF6.

Despite these important strengths, the manuscript presents a significant weakness, namely the lack of a clear definition of “cure” intended as an intervention that can achieve its goal via a single or a limited number of administrations. After that, the curative agent does not need to be administered again either because it continues to be available indefinitely or because the outcome of its activity is irreversible. As such, the infected individual can be safely taken off antiretroviral therapy without viral rebound. Any intervention that requires prolonged, indefinite administration of the agent regardless of the frequency (daily, weekly, monthly, yearly, etc.) does not qualify as a cure but rather as a form of therapy. It is important to have this concept clearly stated, because it discriminates between curative and therapeutic agents and interventions.

Based on the definition above, any (or perhaps most) of the small molecule organic compounds described in this manuscript are not curative agents. They can suppress viral expression and/or replication through various mechanisms. However, they are very unlikely to yield permanent or even long-term effects, and once they are cleared from the body, their effects would be lost. As such, they are more precisely antiretrovirals.

In addition, many of the inhibitors or modulators discussed in this review target host factors. A short-term impact on their function may not cause significant toxicity. This is why the use of HDAC inhibitors and other latency reversal agents in shock-and-kill strategies is acceptable: their activity is only required for a short time span sufficient to reactivate HIV. However, in the context of block-and-lock strategies, drugs that target host factors must have permanent effects (whether or not they are administered repeatedly). Altering the activity of host factors permanently is certainly not advisable. The authors do a great job at highlighting toxic effects that have been experimentally verified or that can be predicted. Nevertheless, any intervention that targets the function of host factors must not be considered for cure strategies. Even genome editing of CCR5 is likely to have detrimental effects. For instance, CCR5 is required for effective immune responses against West Nile Virus and Zika Virus, which are absent in Northern European countries (where the delta32 CCR5 mutant is found) but are present in many African countries where the majority of HIV-infected people live. Of course, this is not the case of drugs targeting viral factors (i.e., dCA or lenacapavir). Yet, their clearance over time does not qualify them as curative agents, but more precisely as long-acting antiretroviral agents that require periodic administration.

A fundamental limitation of many of the drugs discussed in the manuscript is that they also must be available at the appropriate active concentrations at the right time, in most cases during the viral replication cycle. Administration of the drugs when the virus is not actively replicating (for instance during the brief window of viral suppression after ART interruption) does not provide any benefit. For instance, the use of LEDGF inhibitors and CPSF6 modulators to redirect the PIC for integration in transcriptionally inactive regions of the chromatin requires these drugs to be available at the time HIV-1 enters new target cells. How can that be predicted? Even in the case of planned ART treatment interruption, only a small fraction of the existing reservoir (replication competent or now) is reactivated. Therefore, the majority of the existing reservoir continues to be a ticking bomb. To ensure availability of the drugs at all times, they must be administered periodically at a frequency dictated by their pharmacokinetics.

These requirements do not qualify many of the drugs discussed in the manuscript as block-and-lock curative agents. That is, unless we envision block-and-lock as an approach to suppress the viral replication cycle, much the same way as existing antiretroviral drugs already do very effectively and with the critical advantage of targeting viral factors.

The manuscript also lacks additional important definitions of those interventions that can be considered a cure: safety, efficacy, durability, specificity, affordability, and scalability. These are essential requirements, because failure to achieve any of them would essentially disqualify an intervention as curative.

Finally, the manuscript omits to discuss the block-and-lock curative strategies aiming to achieve permanent, epigenetic viral silencing via host and viral long noncoding RNAs, such as MALAT, NEAL, and the HIV antisense RNA. The use of retroviral/lentiviral vectors can ensure their stable overexpression via a limited number of administrations. The use of innovative pseudotyping strategies can achieve cell-specific delivery.

Author Response

Responses to Reviewer 2

Comments 1: In their manuscript, Tolomeo and Cascio discuss current approaches to achieve an HIV cure via permanent silencing of the provirus, collectively known as block-and-lock. This is a major strength of the manuscript because the block-and-lock strategy remains underexplored, and this review shines new light on this alternative approaches. Additional strengths of the manuscript are the breadth and depth of the topics discussed. The authors cover a very broad range of potential host and viral factors that could be the target of block-and-lock strategies. They provide very detailed description of the functional role of each factor, potential approaches to target them along with a thoughtful analysis of their strengths and weaknesses. The authors focus in particular on strategies targeting the host factors LEDGF/p75 and CPSF6.

Response 1: We thank the Reviewer for this important and insightful comment. We fully agree on the need to clearly distinguish between curative and therapeutic interventions in the context of HIV-1 infection. Accordingly, the Introduction has been substantially revised to explicitly incorporate the Reviewer’s definition of “cure.” We have now clearly stated that a curative intervention is one that achieves durable viral control or eradication following a single or limited course of treatment, without the need for ongoing therapy and in the absence of viral rebound after treatment interruption. In contrast, we explicitly define interventions requiring continuous or repeated administration as therapeutic rather than curative.

In line with this clarification, we have revised the text to more critically position current “block-and-lock” strategies. We now emphasize that, although these compounds are widely described as block-and-lock agents, their effects are generally dependent on continued exposure, and their inability to establish a definitive and irreversible “lock” currently limits their classification as curative interventions. This point has been explicitly added in the revised Introduction. Furthermore, we have acknowledged the limited availability of in vivo and clinical data supporting long-term efficacy. Finally, we expanded the conceptual framework of the Introduction by proposing that strategies capable of redirecting proviral integration toward transcriptionally silent chromatin regions may, over time, promote the enrichment of a deeply latent and transcriptionally inactive reservoir. We clarify that this represents a hypothetical mechanism that could, in principle, approximate a functional cure, although this remains to be demonstrated.

Comments 2: In addition, many of the inhibitors or modulators discussed in this review target host factors. A short-term impact on their function may not cause significant toxicity. This is why the use of HDAC inhibitors and other latency reversal agents in shock-and-kill strategies is acceptable: their activity is only required for a short time span sufficient to reactivate HIV. However, in the context of block-and-lock strategies, drugs that target host factors must have permanent effects (whether or not they are administered repeatedly). Altering the activity of host factors permanently is certainly not advisable. The authors do a great job at highlighting toxic effects that have been experimentally verified or that can be predicted. Nevertheless, any intervention that targets the function of host factors must not be considered for cure strategies. Even genome editing of CCR5 is likely to have detrimental effects. For instance, CCR5 is required for effective immune responses against West Nile Virus and Zika Virus, which are absent in Northern European countries (where the delta32 CCR5 mutant is found) but are present in many African countries where the majority of HIV-infected people live. Of course, this is not the case of drugs targeting viral factors (i.e., dCA or lenacapavir). Yet, their clearance over time does not qualify them as curative agents, but more precisely as long-acting antiretroviral agents that require periodic administration.

Response 2: We agree with the Reviewer that targeting host factors raises critical safety concerns, particularly in the context of curative strategies. Accordingly, we have revised the Introduction and Conclusions to explicitly state that approaches requiring sustained or irreversible modulation of host factor activity are unlikely to be compatible with a cure and should be more appropriately considered as therapeutic antiretroviral strategies. A distinct and theoretically interesting case is represented by CPSF6. In this scenario, the combination of two factors—the ability of capsid inhibitors to bias HIV-1 integration toward LADs, and the gradual selection over time of cells harboring proviruses integrated in these transcriptionally repressive regions due to viral cytopathic effects and immune pressure—could, in principle, generate a situation analogous to elite controllers. Integration in highly repressive heterochromatin combined with natural selective pressures could over time promote durable viral silencing, potentially approaching a functional cure. In such a case, cessation of capsid inhibitor treatment might not result in viral rebound. Currently, this concept remains entirely theoretical and requires experimental validation, particularly as capsid inhibitors have only recently entered clinical use.

Comments 3: A fundamental limitation of many of the drugs discussed in the manuscript is that they also must be available at the appropriate active concentrations at the right time, in most cases during the viral replication cycle. Administration of the drugs when the virus is not actively replicating (for instance during the brief window of viral suppression after ART interruption) does not provide any benefit. For instance, the use of LEDGF inhibitors and CPSF6 modulators to redirect the PIC for integration in transcriptionally inactive regions of the chromatin requires these drugs to be available at the time HIV-1 enters new target cells. How can that be predicted? Even in the case of planned ART treatment interruption, only a small fraction of the existing reservoir (replication competent or now) is reactivated. Therefore, the majority of the existing reservoir continues to be a ticking bomb. To ensure availability of the drugs at all times, they must be administered periodically at a frequency dictated by their pharmacokinetics. These requirements do not qualify many of the drugs discussed in the manuscript as block-and-lock curative agents. That is, unless we envision block-and-lock as an approach to suppress the viral replication cycle, much the same way as existing antiretroviral drugs already do very effectively and with the critical advantage of targeting viral factors.

Response 3: We agree with the Reviewer’s point, and we have clearly highlighted this limitation in the Conclusions of the revised manuscript. Specifically, we acknowledge that compounds such as LEDGF inhibitors and CPSF6 modulators require presence at the time of viral entry and integration to exert their effects, which is difficult to predict given the stochastic and limited reactivation of the viral reservoir. Ensuring such temporal coverage would likely require sustained or repeated administration, which limits the classification of these agents as truly curative. However, when discussing the potential, albeit theoretical, long-term effects of integration modulators, the parameters of time and natural selection, as reported in “Response 2” must be considered. As noted in the manuscript, after years of cART treatment, the majority—if not all—of proviruses are integrated into KRAB-ZNF genes, likely reflecting natural selection over time. Despite residing in KRAB-ZNF heterochromatin, these proviruses can reactivate, and upon cART interruption, viral rebound occurs. The open question, and a future challenge, is what might happen if similar selection occurs over time under the pressure of compounds capable of directing proviral integration into highly repressive chromatin. This scenario could, in principle, lead to a reservoir that is more deeply silenced, approaching a state of functional cure, but experimental validation will be required.

Comments 4: The manuscript also lacks additional important definitions of those interventions that can be considered a cure: safety, efficacy, durability, specificity, affordability, and scalability. These are essential requirements, because failure to achieve any of them would essentially disqualify an intervention as curative.

Response 4: In response to the Reviewer’s comment, the revised manuscript, we have clarified that, in addition to safety, efficacy, durability, and specificity (already discussed in detail), affordability and scalability represent critical translational requirements for any intervention to be realistically considered curative at the population level. However, we respectfully note that, for most of the compounds discussed within the “block-and-lock” framework, including transcriptional inhibitors, epigenetic modulators, and integration-targeting agents, there are currently no available data regarding production costs, pricing, or large-scale manufacturing feasibility. These agents are still in preclinical or early clinical development, and therefore any discussion on affordability would be highly speculative. The only partial exception is the capsid inhibitor lenacapavir, which has been approved for clinical use. Nevertheless, its pricing and accessibility vary significantly depending on national healthcare systems. For instance, in Italy, lenacapavir is expected to be provided free of charge to eligible patients through the national healthcare system, making direct cost comparisons or generalizations difficult. In light of these limitations, we have added a statement in the “Conclusions” section acknowledging affordability and scalability as essential criteria for curative strategies, while also emphasizing the current lack of robust data addressing these aspects for most “block-and-lock” agents. We believe this addition appropriately addresses the Reviewer’s concern without introducing unsupported or speculative conclusions.

Comments 5: Finally, the manuscript omits to discuss the block-and-lock curative strategies aiming to achieve permanent, epigenetic viral silencing via host and viral long noncoding RNAs, such as MALAT, NEAL, and the HIV antisense RNA. The use of retroviral/lentiviral vectors can ensure their stable overexpression via a limited number of administrations. The use of innovative pseudotyping strategies can achieve cell-specific delivery.

Response 5: We thank the Reviewer for this valuable comment. In the revised manuscript, we have added dedicated sections addressing long noncoding RNA (lncRNA)-based block-and-lock strategies. We discuss host lncRNAs such as MALAT1 and NEAT1, highlighting their opposing roles in HIV-1 transcription and their potential modulation to promote viral silencing. We have also included a new section on HIV-1 antisense RNA (AST/ASP), summarizing evidence that it promotes durable latency through PRC2 recruitment, chromatin remodeling, and transcriptional interference, and emphasizing its potential as a block-and-lock agent. Finally, we have incorporated a discussion on delivery approaches, including lentiviral vector–mediated stable expression and advanced pseudotyping strategies for cell-specific targeting of HIV reservoir cells. These additions address the Reviewer’s concern and expand the manuscript to include this emerging class of epigenetic silencing strategies.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All of this reviewers concerns have been addressed.

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors have done an outstanding job in revising the manuscript. We are glad that they concurred with the need to distinguish therapeutic vs. curative interventions, as well as warn about possible toxicities and side effects. We are also pleased with the choice of discussing additional curative interventions that had been overlooked in the original manuscript.