The Known Unknowns: Missing Pieces in in vivo Models of Fragile X Syndrome

Pushkar Kulkarni*, Aarti Sevilimedu*

Centre for Innovation in Molecular and Pharmaceutical Sciences (CIMPS), Dr. Reddy's Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad, Telangana, 500046, India

Fragile X Syndrome (FXS) is a rare disease and the leading monogenic cause of Autism Spectrum Disorders (ASD). It is caused by the silencing of the Fragile X mental retardation (FMR1) gene and the subsequent reduction or loss of fragile X mental retardation protein (FMRP). The clinical effects seen in FXS patients are several and highly variable making it difficult to model them in a single model or even one organism. Furthermore, several human behaviours can be measured only through surrogate endpoints in animals. Therefore, it has been challenging to develop in vivo models of FXS for drug discovery.

This review endeavours to consolidate the information on all available in vivo models for FXS specifically with a focus on their suitability for drug development, with the objective of identifying gaps and potential solutions. To do so, we have summarised the major clinical characteristics and possible mechanisms underlying clinical phenotypes associated with FXS and other disorders that arise from abnormalities in the FMR1 locus, such as fragile-X associated tremor/ataxia syndrome (FXTAS), fragile-X-associated neuropsychiatric disorders (FXAND) including ASD and fragile x-associated primary ovarian insufficiency (FXPOI). We then connect clinical features to phenotypes observed in available in vivo FXS models where possible, covering a wide range of organisms from primates, rats, mice, zebrafish, fruit fly and zebra finches. For each model organism, we list the technology of model creation, phenotypes/assays, mechanistic basis of disease manifestation and specific advantages or disadvantages of the model in the context of drug discovery.

Finally, we have highlighted the missing pieces in FXS modelling and propose strategies to address them, considering aspects of modelling spectrum disorders, repeat expansion and silencing, new functions of FMRP and identification of efficacious treatments.


Figure: Missing pieces in modelling of Fragile X Syndrome

Fragile X Syndrome (FXS) is one of the most studied monogenic neurological syndromes over the past few decades. It is caused by silencing of the Fragile X mental retardation (FMR1) gene and the subsequent reduction or loss of FMR protein (FMRP). CGG repeat expansion in the 5’UTR of the FMR1 gene, followed by hypermethylation of the region is the basis of the observed silencing in FXS1. This event occurs sequentially in successive generations, beginning with small repeat expansion (55-200) causing pre-mutation in one generation with a toxic gain of function in the mRNA, followed by further expansion (>200) to full mutation in subsequent generations with complete silencing of the gene2. FMRP is an RNA binding protein3, a well-known regulator of translation, and is known to interact with well over 800mRNAs in the adult neuron4. Even though several of the mRNA targets have not been fully characterised, it can be said that, FMRP loss has a cascading effect on several pathways which result in the observed clinical features5,6. Several model organisms have been used to model this disease, as discussed extensively in the following tables. The major challenges in modelling FXS are listed here (and substantiated in the rest of the review article):

a) The clinical presentation of the disease in humans is highly variable with different individuals showing different sets of clinical phenotypes.

b) Similar to other neurological diseases, the biochemical and pathological profiling of FXS in real time is practically impossible. This makes it extremely difficult to decipher the human pathobiology making it necessary to have in vivo models.

c) The human nervous system is the most evolved and mimicking it in lower mammals and other vertebrates is done only by using surrogate endpoints especially for cognitive and social behaviours.

d) Animal models show high variability in measurable phenotypes.

Therefore, modelling a complex neurological syndrome like FXS is a continuing process and will require a set of organisms to model all the clinical characteristics, to study various pathobiological aspects and to screen potential drug candidates.

Modelling most diseases and syndromes is necessary in disciplines of disease biology and drug discovery; however, given FXS’s monogenic aetiology, these models can be potentially studied for understanding several other aspects including (but not limited to):

a) Pathways of development of various cognitive abilities

b) Specific connective tissue organogenesis

c) Pathways of behaviours like anxiety, depression, irritability, others.

Thus, modelling FXS and improving the models can have great value to the field of neurosciences. In the present review, we discuss clinical presentations and possible mechanisms, various in vivo models and their advantages and disadvantages, and propose the next set of models and methods that can be used to plug in the missing pieces.

Clinical characteristics that are most commonly noticed in patients with FXS and the suggested mechanisms have been presented below in Table 1.


Clinical Presentations

Suggested Mechanisms of Pathobiology


Fragile X-associated neuropsychiatric disorders (FXAND)

Attention-Deficit Hyperactivity Disorder (ADHD)

Anxiety Autism Spectrum Disorder (ASD)

Developmental Intellectual Disability

Language Deficits

Seizures (rarely)

Dysregulation of excitatory and inhibitory neurotransmission by:

  1. mGluR1 mGluR5 (glutamatergic) enhanced signal transduction
  2. Deficits in GABA signalling


Indirect glutamatergic mechanisms that modulate mGluR:

  1. Dysregulation of N-methyl-D-aspartate receptor (NMDAR)
  2. Altered expression, trafficking, and functions of Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs)







Fragile-X associated tremor/ataxia syndrome (FXTAS)

Excess mRNA from FMR1 premutation may lead to FXTAS in the progeny through the following mechanisms:

  1. Toxic FMR polyG protein production
  2. Formation of ubiquitin-positive inclusion bodies through protein & RNA sequestration
  3. DNA damage due to R-loop formation

Its predicted that CCG repeats may lead to sequestration of specific RNA-binding proteins such as lamin A/C, Pura, hnRNP, Sam68, and drosha, thus affecting some of the normal cell function in both FXTAS and FXPOI.

[9], [10]





Fragile X-Associated Primary Ovarian Insufficiency (FXPOI)

Repeat-associated non-AUG (RAN)-translated poly-glycine species (FMR polyG) leading to intracellular inclusions bodies affecting ovarian function.

There is an overlap of mechanisms suggested for FXTAS and FXPOI.

[11], [12]  





Spine abnormalities

FMRP loss-related dendritic abnormalities due to the following:

  1. FMRP2-Cofilin pathway disruption
  2. Matrix metalloproteinase 9 (MMP9) upregulation
  3. Excess soluble amyloid precursor protein (APP) levels

[5], [13], [14]





Increase in hydrophilic glycoprotein granules causing testicular edema followed by scattered collagen fibres and bundles of microfibril. Exact pathway has not been elucidated but may be attributed to connective tissue dysregulation.

[15], [16]




Other Physical Characteristics


Long Face

Prominent Ears

Prominent Jaw

Flat Feet

Joint Hypermobility

Loss of FMRP can lead to dysregulation of the following extracellular matrix and connective tissue components:

  1. Elastin downregulation
  2. Bone morphogenic protein receptor 2 (BMPR2) downregulation
  3. Matrix metalloproteinases (MMPs)upregulation

[16], [5]

Various in vivo models of FXS have been described in Table 2. We have also provided the potential advantages and disadvantages of each of these models.


Species, Strain, Model

Technology used to create model










Non-human primate, Macaca mulatta, Spontaneous

None, analysis from a population of macaque

Not measured

Not evaluated.
Sequencing of 

FMR1 alleles carried out to assess CAG/CGG repeat distribution


CGG repeat distribution and propensity for expansion most similar to that in humans;
Model most suited for behaviour studies

Instance of FXS was not observed;
No further studies have been reported using macaques





Sprague Dawley, Fmr1 em1Sage

Zinc-finger-nuclease mediated knockout of Fmr1 (exon 8) in SD or LEH background

Macroorchidism, alterations in density of dendritic spines; Impaired associative learning and memory.

Loss of FMRP, altered translation profile and development of prefrontal cortex circuitry, altered mGluR5 signalling


Repeated, longitudinal testing of recognition memory paradigms during development;
Identification of suitable treatment window for therapeutics;
Differentiation between preventive and corrective MoA of a given drug

Rat models do not offer significant advantages over murine models with regard to   behavioural phenotypes of FXS.
Genetic background contributes significantly to variability in phenotypes

LEH, Fmr1em1/PWC

Zinc-finger-nuclease mediated knockout of Fmr1 in SD or LEH background


Sprague Dawley, Fmr1 exon4 KO

CRISPR-Cas9, exon 4 mutation leading to truncation

Macroorchidism, altered LTP, LTD, spatial learning and memory; impaired social interaction (novelty recognition)

Loss of FMRP, altered translation and synaptic plasticity


Clear evidence of deficits in hippocampus-dependent spatial learning and memory (unlike murine models)





Knock-outs: C57BL6/J or FVB/N, Sighted FVB, Fmr1tm1Cgr

Homologous recombination with disruption in exon 5

Mild facial dysmorphism;
Susceptibility to seizures;
Neuroanatomical as well as functional brain abnormalities (reduced dendritic spine density, cognitive and behavioural deficits, altered LTD and LTP)

Loss of FMRP, altered translation profile and altered mGluR5 signalling

[23]–[25] and others, summarized in [26]


Most commonly used model;
Very well studied and shows most of the clinical phenotypes.

Residual Fmr1 mRNA present, repeat expansion and promoter methylation absent;
Behavioural and cognitive deficits are mild, social behaviour modelling difficult;
Genetic background contributes significantly to variability in phenotypes.

Conditional KOs: Sighted FVB or C57BL/6J, Fmr1 cON, CKO, KO2

Cre-lox recombination with disruptions in exon/intron 1


Allows tissue or brain region specific function of FMRP (such as in the hippocampus, CA1 neurons etc) as well as temporal regulation (as yet unreported).

Not very widely used, offers no specific advantages over the KO models for general behavioural tests.

Point mutation: C57BL6/J and FVB/N, Fmr1I304N

Cre-lox recombination

Phenocopies KO models

Missense mutation in the KH domain of FMRP prevents RNA as well as ribosome binding, leads to reduced FMRP levels.


Additional model with a different genotype, same phenotype, for drug discovery;
Absence of the neomycin cassette (present in the KOs) provides a minimally altered genome/transcriptome

Models a very rarely observed patient mutation.
Offers no significant advantages over the KO models.

Rescue models: C57BL6/J Fmr1 or Fmr1KO TG298, Fmr1 TG296

YAC transgenesis of human FMR1alleles with 20/92 repeats, into WT

Altered anxiety related phenotypes contrary to those observed in FXS KO

Overexpression of FMRP (~10 fold over endogenous);
Presence of multiple copies of a transgene.


Reveals phenotypes associated with FMRP overexpression;
Useful to study gene therapy and titrate expression for optimal phenotypic rescue;
Provides evidence for rescue of mouse phenotype by human FMRP.

No major repeat expansion observed;
Only partial rescue of phenotypes observed, mechanism unclear.

YAC transgenesis of human FMR1alleles with 20/92 repeats, into Fmr1 KO mice

Rescue of macroorchidism, and anxiety related phenotypes

Rescue of FMRP deficit, with overexpression of FMRP;
Small repeat expansions upon transmission.


Knock In/Repeat Expansion C57BL6/J or FVB/N, Fmr1tm2Cgr

Homologous recombination

Impaired locomotion, altered anxiety, spatial memory and learning deficits

Moderate repeat expansion and instability, leading to reduced FMRP and increased Fmr1 mRNA, intranuclear inclusions in the neurons and astrocytes

Summarized in [31]

Only model to study mechanisms of pathogenic repeat expansion similar to that observed in humans with FXS premutation

Repeat expansion is not accompanied by promoter methylation and silencing, even though FMRP level is reduced;
Not useful for screening methylation-modulation therapies.





Knockdown (transient)


changes in the brain, altered neurite morphology, neural crest specification defects

Reduction in Fmrp, increased mGluR5, perturbed calcium distribution and signalling


Simple, robust knockdown produces strong morphological changes;
Rescued by pharmacological inhibition of mGluR5;
suited for high throughput screening

Morpholino based induction of artefacts may confound some findings;
No behavioural studies.

fmr1hu2787 (stop); fmr1hu2898 (splice)

ENU, (N-ethyl-N-nitrosourea) induced mutations and screening for fmr1knock out

No anatomical defects.
Anxiolytic behaviour, hyperactivity, impaired inhibitory avoidance, altered synaptic plasticity (LTP and LTD)

Loss of Fmrp, increased mGLuR5


Loss of function model illustrates possible mechanism. Demonstrates social deficits.

Mild phenotypes, not suitable for screening. Potential compensatory mechanisms in play.

fmr1-/- mutant


Craniofacial abnormalities, Hyperactivity, Memory deficit

Loss of Fmrp, changes in the expression of genes involved in learning and memory and cartilage development.


First targeted knockout model showing phenotypes similar to observed human FXS symptoms.
Shows rescue with fmr1 mRNA;
Suited for drug screening and longitudinal studies

Mild phenotypes are observed, potentially due to compensatory mechanisms;
Social deficits and other behavioural phenotypes not yet elucidated.

Knock down (transient)


Craniofacial abnormalities,
Anxiety, Attention deficit,

Reduction in Fmrp, increased mGLuR5, altered p-ERK and p-Akt

[37], [38]

Simple, inexpensive transient model for drug screening;
Reveals the effect of limited loss of Fmrp during early development on later phenotypes

Transient knock down. Social deficits cannot be appreciated in short duration knock down.





dfmr1 mutant

P-element insertion

Abnormalities in behaviour, including erratic activity, disrupted sleep patterns, circadian clock; defects in synaptogenesis and spermatogenesis; defects in learning and memory (olfactory, courtship assays)

Loss of Fmrp, impacting glutamate signalling (mGluR5) DNA damage response (DDR), RNA editing (ADAR) and microRNA pathways (Ago1)

[39], [40]

Simple model recapitulating various human and mouse FXS phenotypes;
Powerful forward and reverse genetic approaches to identify new pathway and targets, amenable to pharmacological screens

Promoter methylation and silencing is not modelled;
Evolutionarily most distant, therefore may be least suited to screen for drugs.

Round worm, Caenorhabditis Elegans


Expression of varying CGG repeat length containing reporters.

Loss of olfactory adaptation due to a repeat-associated dysfunction

Repeat containing RNA acts through miRNA-binding Argonaute ALG-2, to impact adaptive olfactory response (a readout for reduced neuronal plasticity)


Potential model to study olfactory plasticity and subversion in neuronal function, and investigate the molecular link between expanded repeats and plasticity in a simplified system.

Only one published report of this model. The reproducibility of this model needs to be established.

Zebra finch, Taeniopyia guttata

No model yet

Vocalization & Language development is dependent on fmr1 expression.

Mechanisms unknown

[42], [43]

Potential model to study the mechanisms behind speech and language pathology.

No model developed so far, feasibility unknown.

Fragile X syndrome is the leading genetic cause of autism, and because of the implication of a single causal gene, animal models of FXS are numerous26. The characteristic clinical manifestation of FXS involves some or all of the following symptoms: long face, macrocephaly, prominent ears, prominent jaw, flat feet, joint hypermobility, macroorchidism (clinical); attention-deficit hyperactivity disorder (ADHD), anxiety autism spectrum disorder (ASD) (psychological), developmental intellectual disability, language deficits (developmental) and strabismus, recurrent otitis, gastrointestinal complaints, obesity and seizures (less prevalent)44. While the various models capture a subset of these phenotypes (see table 2 above), no single model has been able to mimic the spectrum of symptoms and deficits seen in human FXS patients, and this has severely impacted screening and drug discovery efforts. There could be two potential reasons for this:

(i) The first is that the presence, severity and manifestation of FXS symptoms varies widely even in human patients44, and is likely influenced strongly by the genetic background and environmental factors. Therefore, one can argue that inconsistencies are expected in the models as well.

(ii) The second stems from the nature and function of the FMR protein. FMRP is a regulator of translation and is expressed from very early on during development, which means that when FMRP is silenced, the levels of a number of proteins (several hundred in an adult neuron, for example), and consequently a number of signalling pathways are likely to be altered, and differentially so at various stages during development45. The phenotypes observed are a result of these cumulative changes. Even though FMRP per se is fairly well conserved in all of the models, varying degrees of conservation across the target proteins, pathways and differentiation paradigms are likely to cause inconsistencies in phenotypes observed in the models. Restricting discovery programs to one model organism, or picking a single genetic background in a model, is unlikely to be beneficial, since heterogeneity is a feature of the disease, not the model. Therefore, we would like to propose that diversification, both in terms of model organisms and genetic backgrounds may be key to identifying robust and efficacious therapeutic interventions. Focussing on simpler genetic models like Zebrafish and Drosophila, may allow one to conduct high throughput screens in a genetically diverse population, and generate statistically significant results.

In FXS, a repeat expansion in the 5’ UTR of the human FMR1 gene leads to hypermethylation and silencing, which results in a drastic reduction of FMRP. Therefore, control at the level of repeat expansion or methylation are the best therapeutic avenues. In humans, the FMR1 locus naturally contains 6-55 CGG repeats; however, all of the model organisms (except the primate) contain very few repeats, if any. The mechanism of repeat expansion involves different stages in which the premutation stage (55-200 repeats) is associated with an increase in FMR1 RNA levels (toxic gain of function), which may be a prerequisite for the progression to full expansion (>200 repeats) leading to hypermethylation and subsequent silencing. Therefore, lack of a critical number of repeats at the outset, combined with a lack of progressive expansion could explain why the knock-in models do not show any promoter methylation-dependent silencing. With a carefully chosen combination of artificial repeat insertion, control of FMR1 RNA levels and manipulation of the DNA replication or repair pathways to promote slippage, it may be possible to develop such a model. We believe that such studies can be conducted in simpler models like yeast46, used to derive optimal conditions which may then be moved into higher models like Zebrafish and mouse, to create true FXS models. Interventions which aim to interfere with repeat expansion, or those that revert the hypermethylation-driven silencing (small molecules47-49, or genetic interventions50,51) can then be screened in such a model, and will either significantly ameliorate the disease (even a two-fold increase in FMRP level is associated with a significantly higher IQ52) or better still, prevent it.

Since FXS is primarily seen as a disease of the brain, studies have focussed on FMRP’s neuronal function and neuronal phenotypes in the models. FMRP is ubiquitously expressed during early developmental stages, and it is increasingly clear that FMRP interacts with and regulates diverse cellular pathways in addition to its primary function as a translation regulator in neurons. These include regulation of RNA editing and splicing, chromatin structure, cellular differentiation kinetics, ion channel regulation and microRNA pathways53. Therefore, exploring the molecular mechanisms underlying the contribution of these pathways to disease progression or phenotypes could be an important new avenue of research. Traditional translation targets of FMRP may also be influenced by disruptions in these other pathways (for example, AMPA receptor and RNA editing54, microRNA and ion channels55) and may be better rescued by novel treatments or combination therapies.

While molecular mechanisms of FXS are fairly well understood, and small molecules targeting at least ten different pathways are able to rescue several molecular phenotypes such as protein synthesis, synaptic plasticity and calcium regulation, none of these have translated into clinical efficacy in humans56. While one obvious explanation is that this is due to the complexity of, and our lack of understanding of the human system, there are alternative explanations which require due consideration. An important take-away from the clinical trials conducted so far, is that tests which measure core phenotypes like behaviour and cognition directly and not through surrogate indications, need to be developed in order to determine the true efficacy of drug treatment. However, there are many avenues for improvement on the preclinical side.

(i) First, newer models which capture the repeat expansion and methylation features should be developed, since targeting these upstream nodes will result in maximal impact (section above).

(ii) Second, varied genetic backgrounds and multiple model organisms should be employed in order to determine the robustness of the phenotypes or treatment being assessed. The molecular phenotypes are highly conserved from flies to human, and the small molecules being considered for clinical trials have been identified based on these conserved pathways. It may be prudent therefore to conduct such studies in models like Danio rerio (Zebrafish) where true “wild type” animals (wild caught) can be used (incorporating the genetic diversity present in the natural world), with as large a sample size as required to power the statistics. Such an approach may allow one to incorporate the varied baselines in the population (for example, the median increase in anxiety in wild-caught vs. lab-wild type strains50) and multi-factorial influences on the neurological phenotypes during the screening process to make results more robust57. Techniques for simple, rapid and inexpensive model creation, such as transient knockdown of gene expression (using DNAzymes or morpholinos in zebrafish32,37,58, RNAi in D.melanogaster59, and differentiation of patient derived cell lines60) will also aid in increasing the number of varied models available to assess the same phenotype and its treatment. A larger number of treatments (compounds and paradigms) can be tested in a more diverse set of assays in such models, and may drastically improve the chances of finding a drug that will translate well into humans compared to traditional approaches using the mouse model.

(iii) Third, multiple tests or assays which measure the same parameter should be employed in each study, and at least one of these should measure the same parameter as in a clinical trial (such as fMRI or EEG).

(iv) Fourth, drug screening should be conducted in models where the link between the molecular changes due to FMRP loss, to circuitry and behaviour are well-established (such as in olfactory system of D.melanogaster61), and rescue at each stage should be assessed.

(v) Fifth, a number of treatment windows may need to be explored for each class of drug, coupled with longitudinal studies which measure impact over the long term, especially for treatments targeting behaviour. Given that the circuitry can be modulated only in certain windows during development, earlier treatment windows need to be preferentially identified and studied.

(vi) Finally, FMRP appears to play a role in multiple unconnected pathways, therefore genetic studies in models like flies and zebrafish could be used to better understand these new molecular functions of FMRP. Subsequently, combination treatments to address more than a single target at a time may be prioritized for screening.

Given that decades of therapeutic research using the available models of FXS have not led to the identification of a clinically efficacious drug, the possibility that the molecular landscape and regulatory network in the human brain is not sufficiently or completely replicated in any other model, has to be considered for the next phase of therapeutic research. In such a scenario, critical and validated endophenotypes62 may need to be used as a basis for screening directly in a “human” model. Brain organoids satisfy the need for “human origin” as well as provide the genetic, cellular and architectural features of the human brain and could therefore be a powerful platform for identification of critical endophenotypes as well as for screening63. Brain organoids from patient derived cells (hiPSCs) have been used to study varied diseases of the central nervous system64, and are likely to be relevant to FXS, where phenotypes are thought to stem from defective cross-talk between multiple cell types and altered neural circuitry. However, the current state of this technology in terms of the time, skill and expense involved, as well as the inability to sustain organoids in culture to model adult-phenotypes limits its applicability to drug discovery, as on date. Yet another strategy to conduct studies on human origin brain tissue, in an in vivo setting is the clever use of transplanted FXS brain tissue (iPSCs which differentiate after transplantation or neural precursor cells (NPCs)) into the mouse brain65. While the chimeric setting has the same limitations as the mouse model in terms of screening for behavioural or cognitive end points, it is likely to reveal the most authentic, cell-type and microenvironment specific responses to drugs.

Fragile X syndrome is a classic test case for a rare disease model, which despite the availability of numerous models and studies over the decades, has not yielded any therapeutic benefits for patients. We believe that part of the reason for this has been the use of approaches which were developed and standardized on the basis of what has worked for the more prevalent, non-monogenic diseases that have dominated the clinical research and drug development fields. In the case of drug development for rare monogenic disorders where disease manifestation is heavily influenced by the underlying genetic background and treatment needs are primarily symptomatic, radically different approaches like the ones described above may be more fruitful. Implementation of such strategies and the consequent identification of efficacious treatments may cause a paradigm shift in the way rare disease biology, modelling and drug development is practiced in the future.

Work in the authors’ laboratories is supported by funding from Dr.Reddy’s Institute of Life Sciences, Hyderabad, India and Department of Biotechnology (DBT) grant BT/PR28305/GET/119/272/2018 awarded to PK and AS. The authors would like to acknowledge Dr. K. Chatti and Dr. K. Parsa for valuable comments on the manuscript.

The authors have no conflicts of interest to declare.

Intellectual Property Statement: The method for creating zebrafish knock down models using DNAzymes is the intellectual property of Dr. Reddy’s Institute of Life Sciences and is covered by the following patent: Embryonic Zebrafish models using DNAzyme mediated knockdown. Sevilimedu A, Kulkarni P. Application No. PCT/IB2019/051255, Indian Application No. 01741031477. Complete specification filed for Dr. Reddy’s Institute of Life Sciences. The authors are affiliated to Dr. Reddy’s Institute of Life Sciences.

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Article Info

Article Notes

  • Published on: June 26, 2020


  • Fragile

  • Syndrome


Dr. Pushkar Kulkarni
Centre for Innovation in Molecular and Pharmaceutical Sciences (CIMPS), Dr.Reddy's Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad, Telangana, 500046, India
Email: ,


Dr. Aarti Sevilimedu
Centre for Innovation in Molecular and Pharmaceutical Sciences (CIMPS), Dr.Reddy's Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad, Telangana, 500046, India