Monogenic Obesity;

Treatment via Gene-Editing

Biseka De Silva
(CL/HNDBDS/08/22)
 INTRODUCTION

• Obesity = life-long, progressive, life-threatening, genetically-

related, multi-factorial disease of excess fat storage.
• Overweight= BMI ≥ 25 kg/m2
• Obese= BMI ≥ 30 kg/m2
 Prevalence
• Worldwide obesity > doubled
1980

200 million men

• 1.5 billion overweight adults 300 million women
2008

• 43 million overweight children - Rising in developing

2010 <5 years countries at rates > 30%
• 224 million obese/overweight than in developed
2013 children (5-7 years) countries.
• 1.9 billion overweight adults }
650 obese - 1/10 obese adults in the
2016
world
Fig.1: Obesity rates by country in 2021 (WorldPopulationReview,2021)
 Genetics of
Obesity

Syndromic Non-syndromic

Chromosomal
Pleiotropic Monogenic Polygenic
rearrangements

WAGR syndrome MC4R,

Cohen syndrome UCP1
LEP
 MONOGENIC OBESITY
• Single mutation Monogenic non-syndromic obesity:
• Heterogeneous condition set
• Body’s energy homeostasis changes
( food intake & energy expenditure)
Orexigenic = Anorexigenic =
Appetite Appetite
stimulation suppression
Leptin-melanocortin Paraventricular Arcuate Nucleus:
pathway:
Nucleus: neuronal neuronal aggregation
Critical role in
hypothalamic control of band in the anterior of in the mediobasal
food intake hypothalamus hypothalamus
 LEPTIN-MELANOCORTIN PATHWAY LEP-leptin
LEPR-leptin receptor
NPY/AgRP- Neuropeptide Y
Leptin LEPR Agouti Related Protein
POMC-Propiomelanocortin
ARCUATE NUCLEUS
Adipose tissue
- TUB +
+ PC1/3-Prohormone
Convertases 1/3
CPE-Carboxypeptidase E
PC1/3
NPY/AgRP JAK POMC
MSH-Melanocyte

+ Stimulating Hormone
SH2B1 CPE MCR-Melanocortin

- STAT3
Receptor
SIM1-Single Minded
+ 𝛼MSH 𝛽MSH Homolog 1
BDNF-Brain Derived
PARAVENTRICULAR NUCLEUS
MRAP2 Neurotropic Factor
SIM1 TrkB-Tyrosine Kinase
Anorexigenic path: Receptor
MC4R MC3R
Food intake TrkB JAK/STAT3-
JanusKinase/Signal
Energy expenditure Transducer & Activator of
BDNF Orexigenic path:
Transcription
Food intake TUB-Tubby protein

Energy expenditure homolog

Fig.2: Leptin-melanocortin pathway
 SINGLE GENE MUTATIONS
• A single mutation:
LEP • Hyperphagia
LEPR • Diabetes insipidus
POMC
Physical presentations • Hypogonadism
MC4R • Retinal Dystrophy
MC3R • Low heart rate
MRAP2
• hypothyroidism
SIM1
Monogenic obesity
BDNF
NTRK2 • Adrenal insufficiency
SH2B1 • Growth hormone deficiency
KSR2 • High fasting insulin
PCSK1 • Low FSH & LH
Biochemical presentations • Decreased CD4 cells & T-cell
TUB
responsiveness.
LRP2
• Less cortisol
Gene Mutation Inheritance Chromosome Protein Physical presentation Biochemical
location function presentation
LEP Homozygou Autosomal 7q32.1 Anorexigenic Severe hyperphagia, Indetectable/ reduced
s recessive pathway reduced satiety feeling. (bioinactive) leptin.
stimulation. Early-onset obesity Fasting
Orexigenic within one year of age. hyperinsulinemia.
pathway Reduced/normal FSH, LH
inhibition (Proenc et al., 2017).
LEPR Homozygou Autosomal 1p31.3 Leptin receptor Quick weight gain during Decreased/ normal FSH,
s mutation recessive childhood & LH, estradiol,
/ adolescence. testosterone. Reduced
heterozygo Hypogonadotrophic TRH, greater
us hypogonadism. (bioinactive) TSH,
compound Hypothalmic reduced T4 (Proenc et
mutation hypothyroidism with al., 2017).
puberty delay.
MC4R homozygou Autosomal 18q21.3 anorexigenic High bone mass. Greater Hyperinsulinemia. High-
s or co- and orexigenic fat & lean mass. Less risk normal TSH, low-normal
compound /dominant effect of hypertension T4 (Proenc et al., 2017).
heterozygo transmission. although obese.
us or Controls Subclinical
heterozygo satiety and hypothyroidism.
us energy
mutation expenditure.
 JUSTIFICATIONS
• Single point mutationMC4R deficiency; autosomal
dominant inheritance} phenotype always expressed
• Most common monogenic obesity form
• No proper treatment
• Untreated  life-threatening} CHD, Diabetes insipidus
• Social stigma} Humiliated, discriminated  depression
 OBJECTIVE

Verify the feasibility of

Correct the MC4R point
monogenic disease obesity
mutation in vivo & in
treatment by CRISPR/Cas9
vitro using CRISPR/Cas9
technology
technology

H0= gene-editing by rAAV vector would not be a better treatment to correct the MC4R point
mutation
HA= gene-editing by rAAV vector is a better treatment to correct the MC4R point mutation
 Single base
substitution
(C142,CCT-
>TCT)

Homozygous
MC4R Dominant
hereditary
Severe-
Early Onset
dominant
(missense)
POINT disease obesity mutation

MUTATION
Pro substituted
by Ser @ codon
48. Congenital
MC4R deficiency
 MC4R MUTATION ANALYSIS

Endocrine NGS
Hyperphagia, anomalies
Early onset Normal (growth trajectory, confirms
Taller, less risk
severe psychomotor hypothalamic & the MC4R
of pituitary hormone
obesity development point
hypertension testing } insulin,
TSH, T4) mutation
 EXISTING TREATMENTS
Weight loss by
Metreleptin Setmelanotide Lifestyle intervention
Metformin

• LEP • Emerging • Effective • Rational

therapy α-MSH in POMC therapy
• Restores analog & MC4R for
gonadal therapy functional patients
function • Stimulates mutations with KSR2
• Useless in MC4R • Difficult to mutation
LEPR maintain
deficiency
 CRISPR/Cas9 in Gene-Editing
CRISPR/Cas9
RNA guided programmable nuclease
Generates site specific DSBs
Stimulates HR
Allows transgene migration for gene therapy
Recombinant Adeno-Associated Virus
Viral vector derivative of non-pathogenic
ssDNA
Transduce both dividing & non-dividing
cells
Donor vectors for HR in vitro & in vivo

sgRNA
Single guide RNA target site
Reconstituted in the genome after integration
No need to serially transfect & transduce cells
Single step
Intracellular HR machinery naturally iterates the process
Supports high HR rates
Fig. 3: How CRISPR/Cas9 works (Marzo,2019)
 CURRENT TREATMENTS via GENE-DELIVERY
• Targets the liver, striated muscles & CNS
• 145 clinical trials registered @ Clinicaltrials.gov since 2018
Regional rAAV
In commercial use Ongoing trials
distribution
• Luxturna: Spark • Direct • Haemophilia A & B
therapeutics; 1st FDA intraparenchymal (Liver)
approved rAAV drug rAAV • DMD (Muscle cells)
for monogenic vision • SMS (Broad CNS)
injectionsTreat
loss (RPE65 mutation)
CNS diseases. • Huntington’s
• Gylbera: uniQure: disease (Gene
lipoprotein lipase silencing)
deficiency • Lysosomal disorders
 GENE-EDITING of MC4R POINT MUTATION
Recognize sense
Construct 2 Insert into
& antisense
sgRNAs vectors
strands

• High cleavage efficiency of sgRNA-Cas9.

• High specificity of sgRNA recognition
 Design donor to introduce a T>C point mutation.
 Introduce a synonymous mutation in donor’s corresponding
sgRNA site Avoid sgRNA-Cas9 mediated cleavage
 Already approached LEP GENE-EDITING
(in vitro)

Step 1 Identified & isolated primary preadipocytes

• 12h ex vivo culture

Step 2 Identified C>T in exon 2

• Sanger sequencing
 Already approached LEP GENE-EDITING
(in vitro Cont’d…)
Step 3 HDR of an R105X mutation by CRISPR/Cas9

• Control group; avoided false positives due to SNP presence

• Sanger sequencing; verified correct introduction
• T7EI; no off targets
• CRISPR/Cas9; highly active (induced mutations @ frequencies of 75.43+/-1.55%

Step 4 Leptin production significantly restored

• [Leptin] in mature culture supernatant=573.4+/-51.3 pg/ml (higher)
• [Leptin] in control group =30.2+/-1.5 pg/ml
 Already approached LEP GENE-EDITING
(in vivo)
Step 1 LEP mutation corrected via CRISPR/Cas9
• In situ injection to adipose tissue of ob/ob mice (Efficiency >/= 90%)
• TA cloning; HDR efficiency = 1.67%
• Nested PCR; No off-target effects

Step 2 Leptin production restored

• Immunohistochemistry & in situ hybridization; detectable [Leptin] by edited adipose tissue

• H&E staining; no inflammatory / neoplastic changes in adipose tissue due to rAAV infection

• Decrease in body size, bilateral inguinal adipose tissue volume, food intake & weight ⸫ ameliorated obesity.

• Inhibition of TC, TG & LDL-C levels

Improved glucose & lipid metabolism diseases
• Smaller & less weight liver
AdvCas9-sgRNA virus & AdvDonor: highly efficient in
infecting mammalian cells. No diseases / cellular changes

Directly converts one base pair into another

without generating DSBs
Refinement of the DNA base-editor design:
greatly improved editing efficiency, accuracy and
flexibility
Versatile tool in precision medicine, from the functional
confirmation of genomic variants to the development of
therapeutic solutions directed at many genetic diseases
CRISPR/Cas9 system: highly specific, with
no observable off-targets

Radical treatment strategy for monogenic

diseases & ideal for clinical settings with
limited conventional treatments

DNA base editors have an RNA-guided,

catalytically inactive form of Cas: sequence
recognition & a fused effector: achieve base
conversion
 LIMITATIONS
Restriction
Unffordable (high manufacturing cost,
many resources & expertise)
Immunological barriers (E.g. defence
against AAV)
Intracellular trafficking of rAAV involves
multiple cellular events & may abort at
any step causing unsuccessful gene
delivery
Compensation
None (Zhu et al., 2021)
Complete removal of viral coding sequences:
1. maximize the packaging capacity of rAAVs
2. low immunogenicity and cytotoxicity when
delivered in vivo (Zhu et al., 2021)
Identify key cellular host factors & understand
the mechanisms that regulate the process to
improve rAAV transduction efficiency (Zhu et
al., 2021)
 CONCLUSION
• No existing specific treatment for MC4R gene mutation (very abundant)
• Gene-editing is the ideal mechanism to correct the mutation in such patients 
reduces food intake and increases energy expenditure.
• The limitations have been overcome in previous gene-editing treatments.

• Only Luxturna (partial vision obtained) } in human practice (April 2021 in France)

• Not much in human practice } high cost

• Brain injections: impossible ⸫ design a Mab to target PVN

• Reject H0
 ABBREVIATIONS
Abbreviation Term Abbreviation Term
BMI Body Mass Index PC1/3 Proprotein convertase 1/3
LEPR Leptin Receptor CPE Carboxypeptidase E

LEP Leptin MRAP2 Melanocortin 2 receptor Accessory Protein 2

TUB Tubby protein homolog MC3R Melanocortin-3 receptor

NPY/AgRP Neuropeptide Y/ Agouti gene Related Protein MC4R Melanocortin-4 receptor

SH2B1 Src homology 2 B adapter protein 1 SIM1 Single-minded homolog 1

JAK Janus Activated Kinase BDNF Brain-derived neurotrophic factor

STAT3 signal transducer and activator of transcription 3 TrkB Receptor Tyrosin-Kinase B

POMC proopiomelanocortin NTRK2 Neurotrophic tyrosine kynase receptor type 2

MSH melanocyte stimulating hormone TA cloning Rapid cloning

 ABBREVIATIONS
Abbreviation Term Abbreviation Term

KSR2 Kinase suppressor of ras 2 CGH Comparative Genomic Hybridization

PCSK1 Proprotein convertase subtilisin/kexin type 1 NGS Next-Generation Sequencing

LRP2 Low-density lipoprotein receptor 2 LDL-C Low-density lipoprotein cholesterol

FSH Follicle Stimulating Hormone sgRNA single synthetic guide RNA

LH Luteinizing Hormone rAAV Recombinant Adeno-Associated Virus

TRH Thyrotropin-Releasing Hormone DSBs DNA double-strand breaks

TSH Thyroid-stimulating hormone HR Homologous Recombination

T4 Thyroxine PAM protospacer adjacent motif

ACTH Adrenocorticotropic hormone CNS Central Nervous System

 ABBREVIATIONS
Abbreviation Term
FDA Food & Drug Authority

RPE65 Retinal pigment epithelium-specific protein 65kDa

DMD Duchenne muscular dystrophy

SMS Smith-Magenis syndrome

T7EI T7 Endonuclease I

HDR Homology Directed Repair

PCR Polymerase Chain Reaction

TC Total Cholesterol

TG Triglycerides

TrkB Tyrosine kinase receptor

 Ackowledgement

• ICBT City campus

• Mrs. Jayani Kariyawasam
• Mrs. Miruna Rabindrakumar
 REFERENCES
• Bak, R. O. and Porteus, M. H. (2017) ‘CRISPR-Mediated Integration of Large Gene
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10.1016/j.physbeh.2020.113134.
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 REFERENCES
• Proenc, A. C. et al. (2017) ‘PT NU SC’, Journal of Diabetes and Its Complications. Elsevier Inc.
doi: 10.1016/j.jdiacomp.2017.04.026.
• Saeed, S. et al. (2015) ‘Genetic variants in LEP, LEPR, and MC4R explain 30% of severe obesity in
children from a consanguineous population’, Obesity, 23(8), pp. 1687–1695. doi:
10.1002/oby.21142.
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<https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight> [Accessed 3
July 2021].
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By Country 2021 (worldpopulationreview.com)> [Accessed 3 July 2021].
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CRISPR/Cas9 system’, Journal of Genetics and Genomics. Elsevier Limited and Science Press,
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