A novel rhesus macaque model of Huntington’s disease recapitulates key neuropathological changes along with motor and cognitive decline

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1. 

   Version published to 10.7554/elife.77568 on eLife

   Oct 7, 2022
2. 

   eLife

   Aug 16, 2022

   Evaluation Summary:

   The authors show the utility of an AAV-based approach in non-human primates to develop an improved model of Huntington's disease. They have presented a very thorough, carefully executed, body of work that will be of benefit to a range of researchers studying HD or developing therapies for HD. While this extends the work from an earlier paper (that presented the tools used to induce phenotypes) the results presented are new, relevant, and important to the community.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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3. 

   eLife

   Aug 16, 2022

   Reviewer #1 (Public Review):

   Weiss et al have developed a novel model of Huntington's disease (HD) by injecting a mixture of recombinant adeno-associated viral vectors (AAVs) into the caudate and putamen of rhesus macaque monkeys. There is a significant need for relevant models of HD. While many mouse models exist, current models lack genetic relevance (with repeat lengths much longer than those found in humans being used) and mice lack the anatomical relevance to humans since they have small brains with important brain regions (in particular the neostriatum) being dissimilar to those seen in humans. The authors used non-human primates because they have large brains with anatomy similar to humans. They used a mixture of recombinant adeno-associated viral vectors (AAVs) in an attempt to overcome the shortcomings of previous models using … More

   Reviewer #1 (Public Review):

   Weiss et al have developed a novel model of Huntington's disease (HD) by injecting a mixture of recombinant adeno-associated viral vectors (AAVs) into the caudate and putamen of rhesus macaque monkeys. There is a significant need for relevant models of HD. While many mouse models exist, current models lack genetic relevance (with repeat lengths much longer than those found in humans being used) and mice lack the anatomical relevance to humans since they have small brains with important brain regions (in particular the neostriatum) being dissimilar to those seen in humans. The authors used non-human primates because they have large brains with anatomy similar to humans. They used a mixture of recombinant adeno-associated viral vectors (AAVs) in an attempt to overcome the shortcomings of previous models using AAVs. They studied their animals over 20 months using both behavioural tasks and MRI assessment. The animal served as their own controls for the imaging, which improves the power of the study. The methods of analysis, particularly the imaging, are modern and directly relevant to assessments that can be conducted on human patients.

   Strengths
   The major strength of the paper is that the authors used Rhesus macaque, a species that is highly relevant to studies aimed at assessing therapies and drug delivery. As a stepping-stone to humans, the macaque has many advantages, including brain size, relevant anatomy and in particular, longevity compared to mice. The approach of mixing a number of recombinant AAVs is also interesting since it overcomes some of the limitations of individual AAVs as detailed in the Introduction. The tasks used for behavioural assessment to investigate the effect of the AAV on brain and behaviour also highlight the advantages of a monkey model, since human-relevant assessments were used. The study was very well controlled, with both vehicle and AAV containing non-pathogenic length CAG repeat (10Q) used.

   The behavioural assessment was comprehensive, and the motor control measures are relevant to HD. MR imaging is also very relevant to what can be measured in humans. The imaging was comprehensive and of excellent quality. Overall, the study presents some important and interesting data, because an acute monkey model has not been studied in such depth previously. The range of approaches taken to assessing the animals is comprehensive and impressive.

   This is a study that will be of interest to researchers who are developing methods for studying the role of the caudate/putamen in behaviour. I agree with the statement that the model will be useful for studying the effects/causes of disruption to the cortico-basal ganglia. The effect of the lesions on cortical regions of the brain are interesting, and well presented.

   Weaknesses
   The major weakness of the study is that with the interpretation of the results. The changes in tractography, behavior and TBM are what would be expected following lesions of the neostriatum. Indeed, all the data point to this being an acute lesion model, and in my opinion, the authors have made an interesting novel neurotoxic model (using a very relevant neurotoxin). Unfortunately, there is no detailed pathology showing what is happening at the level of the striatum or associated cortical regions (see also below). The results have been interpreted as showing a progressive model, although evidence that there is progression is limited. The whole manuscript is written as though this is a genetically-relevant progressive model of HD. But the animals are normal, and so there is no genetic context relevant to HD. While the authors present this as a new model of HD with progressive motor and cognitive changes (as seen in the title of the paper) there is little evidence presented that there are major 'progressive' changes seen. Furthermore, the idea that the changes mirror those seen in HD patients (as stated in the discussion) is somewhat misleading. While their data may be similar to some findings in patients who have early degenerative changes, there are many differences that are either not seen or not explored in the new model. In particular, evidence that the changes seen in the monkeys are relevant to progression of disease in HD patients is missing. While this appears to be a sophisticated acute lesion model that has been assessed more thoroughly than previous models, nevertheless all evidence points to it being an acute rather than a progressive model. While I agree that the data show clearly that there are changes in motor function and to a lesser extent in cognitive function caused by the injection of the AAVs, it does not follow that the changes seen are 'progressive'. Even in rats following striatal lesions, changes in behaviour have been measured for months afterwards. For example, in acute lesions of monkeys, Deglon et al showed years ago that in NHPs the behavioral sequelae of acute neurotoxic lesions change with time. This model also does not show 'progression' in the sense of a progressive disorder in HD.

   The authors state in the Abstract that the injection resulted in "robust expression of mutant huntingtin in the caudate and putamen". These data are not in the manuscript. This seems to be deduced, rather than measured experimentally.

   A disadvantage of the method used, that has been a bugbear of the field, is that the authors chose to use a fragment of the HD gene, with a very long repeat that is seen only in juvenile patients. While using the fragment rather than the whole gene is a sensible approach, since it is known to be toxic, I am not clear why they chose to use a juvenile length repeat rather than a repeat in the adult-onset pathological range. There are mouse models with a CAG repeat of 40-50, but short lifespan of mice has limited their usefulness. Longevity is one of the major advantages of using a monkey. Had they used a repeat of 45 or 50, this would have been a much more interesting paper, because there is little known about the toxicity of proteins with that length repeat in vivo. As it stands, the model is a non-human primate acute fragment model using a long repeat. The disadvantages of fragment models and long CAG repeats has been well discussed in the literature and is a major criticism of many of the mouse models. Consistent with the disadvantages of fragment models with long repeats, the onset of the symptoms of the monkeys is much more rapid that would be expected in either juvenile or adult-onset HD and is likely to be due to both the fragment nature of the vector and long CAG repeat. The rapid onset of phenotype is not discussed in the context of other models.

   The use of a DA receptor agonist was an interesting idea, because DA agonists have been shown to exacerbate abnormal involuntary movements in HD patients. The mechanism for this is complicated, however, given that the balance of D1 and D2 receptors changes as HD pathology progresses. The authors chose to use a non-selective agonist, which caused transient changes in behaviour. However, the usefulness/relevance of the apomorphine data is unclear, particularly since the effect was only seen at the early timepoints and not at the later timepoints. If the AAV causes acute toxicity, then such changes in response to apomorphine would be expected, and this would be expected to resolve with time - as was seen.

   For their cognitive testing, the authors used a task (delayed non-match to sample) that measures object recognition and familiarity. Before surgery, only 11/17 of the animals were successfully trained to complete this task. It is not clear how useful the data are when only 64% of the animals can be included. It would have been better to have choosen a task that all monkeys could perform at baseline.

   The results of the motor task showed a clear deficit. This would be expected with an acute lesion of the striatum. It was interesting that there was a significant improvement from the 9-month point to the 20-month point in the 85Q lesioned monkeys, whereas the performance of the vehicle-treated monkeys plateaued. There appeared to be a deleterious effect on the 10Q monkey performance that was maintained. (This was not discussed in any detail but should be.) This begs the question as to whether or not the 85Q-lesioned monkeys would recover to a level similar to the 10Q animals if left for another 12 months.

   The tractography and tensor-based morphology data are clear, and consistent with the idea that a Q85 fragment would be neurotoxic. The authors consistently suggest that this is relevant to early stages of HD, but there is little evidence presented to support this statement. The sensitivity of the MRI used shows that multiple regions were affected beyond the lesion sites. This would be expected and is new and interesting data in the non-human primate field, although it does not bring anything particularly new to the table with respect to HD (since similar findings have been shown in lesion studies using mice, rats and sheep).

   For HD researchers investigating aspects of the disease, such as transition from prodromal to early symptomatic stages, or for developing treatments, the usefulness of this model will be limited. It is not clear how this monkey model will be useful for developing either disease biomarkers or therapeutic strategies for HD (as stated in the abstract). For studying biomarkers of the disease, this model lacks a number of critical parameters. First, the genetic context of the disease is missing. Second, it is known that HD has multiple sites of pathology, and that symptoms are not simply due to degeneration of the caudate/putamen and that multiple regions of the brain where mutant Htt is expressed become dysfunctional and eventually degenerate. Understanding how the caudate/putamen degenerate is important, but since last century HD researchers have been very aware that dysfunction in the HD brain occurs at many sites other than the caudate/putamen. Indeed, it is probably not only a brain disease since there is evidence of peripheral pathology in humans and other models.

   The authors state that they hope the model will become a widely used resource. This seems an unlikely scenario, given the limitations of the current study and the challenges associated with using monkeys. They say that a major advantage of their technique is being able to generate large numbers of monkeys. But this is not a relevant argument if the usefulness of the model to investigate HD is not proven. Studying the role of the caudate/putamen in motor behaviour is interesting for a small field but limited in scope.

   The authors suggested a number of experiments that could be done, for example, using a shorter HD-relevant CAG repeat length. But as stated above, this is a weakness of the current study, and it would be much more useful had the authors done this experiment themselves. It seems unlikely that until the authors prove its usefulness, this model will not become a widely used resource, since the disadvantages of the model outweigh the advantages. Using monkeys requires a specialist laboratory and facilities and a careful consideration of the ethics involved in animal experimentation. Unless the model offers clear advantages over other models, it is unlikely to become mainstream. It is also not clear what therapies could be tested in this model that could not be tested in other existing models. For example, given that there is no control over which cells are infected by the AAVs, or if any of the cortical pathology is due to spread of AAV from the initial sites of injections, it is not clear how antisense oligonucleotides efficacy could be tested.

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4. 

   eLife

   Aug 16, 2022

   Reviewer #2 (Public Review):

   The authors show the utility of an AAV-based approach in NHPs to develop an improved model of Huntington's disease. They have presented a very thorough, carefully executed, body of work that will be of benefit to a range of researchers studying HD or developing therapies for HD. While this extends the work from an earlier paper (that presented the tools used to induce phenotypes) the results presented are new, relevant, and important to the community.

   The major strengths of the work are the careful assessments done on the animals and the comparators to known human data in HD patients. This includes the behavior testing and the imaging. The data support the conclusions as presented. The major weaknesses are the manner in which the data is presented - it is very hard for the generalist to make sense of many of … More

   Reviewer #2 (Public Review):

   The authors show the utility of an AAV-based approach in NHPs to develop an improved model of Huntington's disease. They have presented a very thorough, carefully executed, body of work that will be of benefit to a range of researchers studying HD or developing therapies for HD. While this extends the work from an earlier paper (that presented the tools used to induce phenotypes) the results presented are new, relevant, and important to the community.

   The major strengths of the work are the careful assessments done on the animals and the comparators to known human data in HD patients. This includes the behavior testing and the imaging. The data support the conclusions as presented. The major weaknesses are the manner in which the data is presented - it is very hard for the generalist to make sense of many of the figures and the written results because they are written as a statistical summary without much context as to the value and novelty of the findings. Also the legends are brief and not very informative, some of the fonts on the figures are extremely tiny and images are crowded, and the colors stated in some figures are not obvious on the figures themselves. These concerns are all fixable.

   The authors would benefit from talking more about their model in the introduction and including references to some key points. For example, there has been critical new data in the field showing the importance of poly (CAG) in disease, not necessarily poly(Q), and the community will want to know (and not be required to look up), the nature of the transgene. Is it a pure CAG repeat? A mixed repeat? If it is pure, do they see or could they measure somatic expansion in the various brain regions impacted? How does that data match the phenotypes seen? Since this is a transgene, there is no possibility for the exon1/intron1 splicing variant to appear - how does this impact their interpretation?
   Also - what about RAN translations? Is RAN translation noted at all in this over-expression model? How does that contribute (or not) to the progressive phenotype they see in their NHPs? These considerations may be more relevant to the community than the comparison/review of the sheep and pig models.

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5. 

   eLife

   Aug 16, 2022

   Author Response

   Reviewer #1 (Public Review):

   1. “The major weakness of the study is that with the interpretation of the results. The changes in tractography, behavior and TBM are what would be expected following lesions of the neostriatum”

   We appreciate this comment and would like to offer clarification. We respectfully disagree that the pattern of results presented in this manuscript are akin to what would be expected following striatal lesions. In NHPs, striatal lesions typically cause more extreme phenotypes than what we observed in our 85Q-treated animals. In macaques, bilateral putamen lesions can result in phenotypes that include seizures, inappetence, hyper-aggression, and other severe features. This strongly impacts clinical scores and can make it unfeasible to care for the animals for multiple years. For these reasons, … More

   Author Response

   Reviewer #1 (Public Review):

   1. “The major weakness of the study is that with the interpretation of the results. The changes in tractography, behavior and TBM are what would be expected following lesions of the neostriatum”

   We appreciate this comment and would like to offer clarification. We respectfully disagree that the pattern of results presented in this manuscript are akin to what would be expected following striatal lesions. In NHPs, striatal lesions typically cause more extreme phenotypes than what we observed in our 85Q-treated animals. In macaques, bilateral putamen lesions can result in phenotypes that include seizures, inappetence, hyper-aggression, and other severe features. This strongly impacts clinical scores and can make it unfeasible to care for the animals for multiple years. For these reasons, recent NHP HD lesion models have used only unilateral putamen lesions coupled with bilateral caudate lesions to model HD (as in the recent paper by Lavisse et al, 2019). Of additional relevance is that even the cognitive effects of these striatal lesions are more severe than what we observed in our 85Q-treated animals: for example, Lavisse reported reduced performance on similar “prefrontal” cognitive tasks by ~50%, whereas our AAV-HTT model exhibited only ~10% reductions in working memory. This mild, but significant, change in cognitive performance and motor function seen in our 85Q animals is much more akin to that which is observed in the early stages of HD.

   2. “The results have been interpreted as showing a progressive model, although evidence that there is progression is limited”...“begs the question as to whether or not the 85Q-lesioned monkeys would recover to a level similar to the 10Q animals if left for another 12 months”

   At the request of Reviewer 1, we added an additional 30-month timepoint and re-ran all of the analyses to include these new data. All of the behavioral and neuroimaging data were re-analyzed with this final timepoint included (see Lines 125-141, 146-163, 173-194, 228-255, 270-294, 314-345). Additionally, due to the unidirectional nature of our hypothesis and on the advice of our bio-statistician, we applied one-tailed tests to the planned comparisons in this revision. To address the Reviewer’s point directly: 85Q-treated animals showed minimal evidence of functional recovery between the 20- and 30-months timepoints on the behavior tasks. In particular, working memory deficits measured with SDR and fine motor skills measured with Lifesaver Retrieval did not improve between 20- and 30-months (Figure 1C and 1F). Additionally, neurological rating scores in group 85Q remained consistently elevated (in the 5-7 range) between the 20- and 30-month timepoint. Taken together, we feel confident that these results do not show evidence of any significant functional recovery, out to 2.5 years (30-months). In terms of the longitudinal trajectories of the behavioral measures, we appreciate the Reviewer’s feedback regarding the use of the term ‘progressive’ and have tempered our language appropriately. We removed all instances of the word progressive/progressed except in the context of the motor rating scores, which show a significant Group x Timepoint interaction and demonstrate a clear progression.

   3. “The whole manuscript is written as though this is a genetically-relevant progressive model of HD. But the animals are normal, and so there is no genetic context relevant to HD”

   We thank Reviewer 1 for this comment. We recognize that viral-based animal models of HD, including the model characterized here, are not as genetically similar to the human condition compared to some of the other modeling approaches currently under investigation (ex. knock-in and gene editing). Limitations of the AAV-based HTT85Q model include: 1) vector packaging restrictions that prohibit expression of full-length HTT, 2) the use of a CAG promoter vs. an endogenous promoter that leads to overexpression of the transgene, 3) the use of cDNA versus genomic DNA excludes introns and therefore lacks the ability to produce alternatively spliced variants (ex, Exon 1), 4) the use of a mixed CAG-CAA repeat may preclude the possibility of somatic instability and 5) expression of HTT that is restricted to specific brain regions and cell types. All of these important limitations have been added to the discussion section in this re-submission (Lines 503-517).

   Despite these limitations, we feel that this AAV2:AAV2.retro-HTT85Q based model has some features that make it genetically-relevant to human HD including: 1) the expression of an N-terminal fragment of human HTT (N171), 2) the N-terminal fragment bears a pathological PolyQ expansion (85Q), 3) the expressed mHTT fragment forms neuronal aggregates that can be detected in the nucleus, 4) mHTT fragments are expressed in many of the same brain regions where aggregates are detected in human HD cases, with both regional and sub-regional specificity (ex. higher expression in anterior vs posterior cortical regions and expression primarily limited to deep cortical layers V/VI) and 5) expression of mHTT fragments in these regions leads to many of the same pathological and behavioral changes observed in HD patients. Importantly, expression of the N-terminal portion of HTT allows for the evaluation of HTT lowering therapeutics that target first 3 exons (ASOs, miRNAs, zinc finger repressors, CRISPR-based therapies, etc), which cannot be evaluated in lesion-based models.

   4. “The authors state in the Abstract that the injection resulted in "robust expression of mutant huntingtin in the caudate and putamen". These data are not in the manuscript.”

   Evidence of mHTT expression in the caudate and putamen, as well as several other brain regions, via immunohistochemical and immunofluorescent staining is now included in the manuscript. Please see additions to the methods, results and discussion sections regarding these findings, as well as a new Figure 5, (see Lines 347-376, 756-788). Additionally, further details regarding an associated PET imaging study in this same cohort of animals using a mHTT aggregate-binding radioligand has been added to the discussion, (see Lines 437-443). Please also see response #13 (below).

   5. “The authors chose to use a fragment of the HD gene, with a very long repeat that is seen only in juvenile patients”

   Comments regarding the need to use a fragment of the HTT gene, versus the full-length gene, due to packaging constraints of the viral vector, were added to the discussion in the context of limitations (Lines 503-517), and also discussed above in response #3. The choice to use a CAG repeat length of 85 (83 pure CAGs followed by a CAA/CAG cassette -see response #17 below for further details), was based off previous studies wherein similar CAG repeat lengths were used to create animal models of HD over the past several decades. Interestingly, while CAG repeat lengths in patients with adult-onset HD typically range from ~40-60, longer repeat lengths (>60) are typically required in animal models of HD to elicit pathological and behavioral manifestations of disease: transgenic, knock-in and viral vector-based rodent models (ranging from 72-150 CAGs), OVT73 transgenic sheep model (73 CAGs), transgenic and knock-in minipig models (ranging from 85-150 CAGs), transgenic and viral vector-based macaque models (ranging from 82-103 CAGs). See Ramaswamy et al, 2007 and Howland et al, 2021 for thorough reviews of these models.

   6. “For their cognitive testing, the authors used a task (delayed non-match to sample) that measures object recognition and familiarity. Before surgery, only 11/17 of the animals were successfully trained to complete this task. It is not clear how useful the data are when only 64% of the animals can be included.”

   We appreciate the Reviewer’s concerns and have decided to conservatively remove this data from the revised manuscript.

   7. “It is not clear how this monkey model will be useful for developing either disease biomarkers or therapeutic strategies for HD (as stated in the abstract)”. “The authors state that they hope the model will become a widely used resource. This seems an unlikely scenario, given the limitations of the current study and the challenges associated with using monkeys. They say that a major advantage of their technique is being able to generate large numbers of monkeys. But this is not a relevant argument if the usefulness of the model to investigate HD is not proven.”

   We thank the reviewer for requesting clarification on these important points. We believe that this model will be useful for developing therapeutic strategies because the HTT85Q-treated macaques express mutant HTT, along with HTT aggregates, in several key brain regions that are affected in human HD, along with undergoing regional gray matter atrophy and white matter microstructural alterations that correlate well with behavioral dysfunction. Studies currently under review elsewhere also show reduced dopamine neurotransmission and regional hypometabolism via PET imaging in this model. Together, or individually, these imaging and behavioral changes can serve as outcome measures when screening potential therapies. Possible therapeutic interventions that are amenable to screening in this model are included in the discussion.

   Regarding biomarker development, we have already engaged in PET imaging biomarker development in this model in collaboration with the CHDI foundation and the Molecular Imaging Center at the University of Antwerp, evaluating a candidate radioligand that binds to aggregated mHTT. See #13 below for a more detailed description of this PET study, including recent data showing its ability to bind to aggregated species of mHTT in several brain regions in this same cohort of HTT85Q macaques that correspond to 2B4 and em48 IHC staining (a manuscript describing these results has been prepared for submission and the PDF is included for the reviewers to peruse).

   The authors do envision this AAV-based macaque model becoming a resource for the HD research community. While this model does have certain limitations (now detailed in the Discussion), we respectfully assert that all of the HD animal models, both small and large, each have their own important limitations to consider when deciding on which to use to screen therapeutics. Selecting a specific animal model based on the individual scientific questions being asked will be required, and employing a combination of models may be an even more prudent strategy.

   While NHP research presents unique challenges (cost, housing requirements and recent challenges in availability, among them), we believe that viral vector-based NHP models could be more accessible to the HD research community compared to some of the other established large animal models; in that they may able to be readily created at contract research organizations (CROs), in addition to various academic research institutions. There are now many CROs that exist in the US, and elsewhere around the world, that have developed specific expertise in MRI-guided, intracranial delivery of AAVs into the NHP brain (including the caudate and putamen), in the context of assessing therapeutic interventions for a variety of neurological disorders (HD, PD, and MSA, among others). Most of these same CROs also have expertise in NHP imaging (MRI/DTI) and behavioral assessments across multiple domains. It seems feasible that AAV-mediated HD macaques could be produced in sufficient numbers to appropriately power therapeutic studies, using the outcome measures established in the current study.

   Reviewer #2 (Public Review):

   1. “The major weaknesses are the manner in which the data is presented”

   We replotted all of the figures with improved color palettes and larger font sizes to make them easier to read. We also added additional details throughout the results section to aid in clarity and improve readability.

   2. “The authors would benefit from talking more about their model in the introduction and including references to some key points. For example, there has been critical new data in the field showing the importance of poly (CAG) in disease, not necessarily poly(Q), and the community will want to know (and not be required to look up), the nature of the transgene. Is it a pure CAG repeat? A mixed repeat? If it is pure, do they see or could they measure somatic expansion in the various brain regions impacted? How does that data match the phenotypes seen? Since this is a transgene, there is no possibility for the exon1/intron1 splicing variant to appear - how does this impact their interpretation”

   Further details regarding the transgene have been added to the Viral Vector Section of the Methods (Lines 531-550). The repeat is not pure and contains a single CAA interruption. The glutamine encoded repeat for HTT85Q contained 83 pure CAG repeats, followed by a single CAA/CAG cassette, while the glutamine encoded sequence for HTT10Q contained 8 pure CAG repeats followed by a single CAA/CAG cassette. Both constructs contained a proline stretch distal to the glutamine repeat in the following allelic conformation where QT represents the total glutamine length:

   HTT85Q: QT=85, (CAG)83(CAACAG)1(CCGCCA)1(CCG)7(CCT)2

   HTT10Q: QT=10, (CAG)8(CAACAG)1(CCGCCA)1(CCG)7(CCT)2

   There are plans to probe for somatic expansion in various brain regions, including the caudate and putamen, as well as several distal cortical regions. That analysis is ongoing and not in the scope of the present manuscript; however, these analyses are now mentioned in the discussion section (lines 540-560), as well as a discussion on the ability to either remove or duplicate the CAA/CAG cassette to potentially increase or decrease the rate of disease progression, respectively, based on the work of Ciosi et al. 2019. Additionally, Reviewer 2 is correct in that the lack of intronic sequences in the transgene precludes the formation of splicing variants, such as the exon1/intron1 variant, which we know is pathological based on the work of Bates et al. This drawback has been added to the discussion, along with other limitations of this viral vector-based model (Lines 503-517).

   3. “What about RAN translations? Is RAN translation noted at all in this over-expression model? How does that contribute (or not) to the progressive phenotype they see in their NHPs?”

   We are also curious regarding the assessment of toxic protein products from RAN translation of the expanded repeat sequence in this model. These studies are planned, and the results of these assays will be included in a future manuscript describing other ongoing post-mortem evaluations in this model.

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6. 

   Version published to 10.1101/2022.02.02.478920v1 on bioRxiv

   Feb 4, 2022