Tрансгенные модели на животных в
трансляционной биомедицине
Рауль Радикович Гайнетдинов
Санкт-Петербургский Государственный Университет, Санкт-Петербург
Сколковский Институт Науки и Технологии (Сколтех), Москва
Итальянский Институт Технологий, Генуя, Италия
Трансляционная медицина
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Трансляционная медицина — это междисциплинарная область знаний,
объединяющая элементы клинической медицины и последних достижений
в молекулярной и клеточной биологии, физиологии, генетики,
биоинформатики, фармакологии, химии, физики и других областей знаний
для максимально быстрой и эффективной разработки и применения новых
терапевтических и диагностических подходов в клинике.
Правительство США через National Institutes of Health в 2012 создало
National Center for Advancing Translational Sciences (NCATS) с бюджетом
более 1 млрд долларов в год, Евросоюз - около 600 млн. евро в год только
через 7 Рамочную программу, Британский Совет по медицинским
исследованиям – более 354 млн. фунтов стерлингов в год и т.п.), фонды и
корпорации (например, фонд Tufts Clinical and Translational Sciences
Institute), медицинские сообщества и т.п.
Только в США открыто более 60 Институтов Трансляционной Медицины в
Университетах (финансируются частично через NCATS)
К сожалению, из опубликованных за последние 5 лет 175 тыс. научных
статей с ключевыми словами «Translational Sciences» только 30 имеют
российскую аффиляцию (по данным Scopus).
Экспериментальные животные в
трансляционной медицине
Выпускник СПБГУ, Нобелевский лауреат Иван Петрович Павлов в
своей лаборатории с экспериментальной собакой
Genomic revolution
HISTORIC ERA
B.C. (Before Christ) – До Нашей Эры
A.D./A.C. (Anno Domini/After Christ) – Нашей Эры
GENOMIC ERA
B.C. (Before Cloning)
A.C. (After Cloning)
Translational Medicine in
Post-Genomic Era
• Humans have about 20,000 genes
• All existing pharmacological drugs currently on
market target approximately 120 proteins
• 100 best selling drugs on the market target 34
proteins
• It is expected that in human genome there are at
least 5000 potential druggable targets
• More or less we understand today functions of
about 10,000 genes
Translational Medicine in
Post-Genomic Era
• Rodents and humans share up to 99% of
genes
• Mice and rats are routinely used in
preclinical pharmacological studies
• Opportunities for genetic manipulations
Opportunities for genetic
manipulations in rodents
• 1982 First Transgenic Mouse
Transgenic animal – one that
carries a foreign gene that has
been deliberately inserted into
its genome.
A team led by Richard Palmiter
created mice expressing a rat
growth-hormone gene.
However:
Generally non-selective expression
of not controlled number of copies
of genes
Opportunities for genetic
manipulations in rodents
• Knockout (KO) mice
• Knockout mutation – replacement of a gene segment by
homologous recombination that normally results in a
nonfunctional or “null” allele.
• First KO mouse line was developed in 1989
Oliver Smithies, Mario R. Capecchi and Sir Martin J. Evans,
won the 2007 Nobel Prize in medicine for developing the
“knockout” technology that allows scientists to create animal
models of human disease in mice
Opportunities for genetic
manipulations in rodents
• Conventional Knockout (KO) mice
Leptin KO mice – model of obesity
However:
• The absence of critical proteins through the development raises
possibility of developmental compensations
• Global disruption of protein expression may result in a complex
phenotype
Conditional knockout mice
• Tissue-specific knockout
– Avoids embryo lethality
– Avoids complex phenotypes
• Inducible knockout
– Allows “before” and “after” type analysis
– Model of acquired mutation rather than
inherited mutation
Other Possibilities for Transgenesis
• Knock-in (KI) mutants – similar to a knockout mutation,
except mutation is usually a point mutation that results in
a partially functional or nonfunctional allele.
• Knock-down (KD) mutants – similar to a knockout
mutation technique used to create a partially functional
allele.
• BAC (Bacterial Artificial Chromosome) transgenic
overexpressors – allows expression of several copies of
a gene and results an increase in protein expression in
endogenous sites.
• Talen, ZFN and CRISPR nucleases technology - the
• most recent opportunity for genome editing in all species
and in shortest time (in mice and rats – 6 months)
Application of transgenic models in
experimental medicine and pharmacology
• Novel target validation by screening for relevant
phenotypes of KO mice
• Testing of hypotheses of disease pathology and
uncovering novel molecular pathological mechanisms
and targets for pharmacological correction
• Recapitulation of human pathology and mutations to
develop naturalistic models of a disease
• Validation of the selectivity of action of novel
compounds
A Mouse for Every Gene
A global initiative to knock out
every mouse gene
NIH Knockout Mouse Project (KOMP) –
about 10,000 strains
Similar consortiums in EU (EUCOMM),
Canada (NORCOMM), China
Lexicon Genetics, Inc.
• Original business model – generate and phenotype KOs
for every gene to build database of phenotypes and
patent portfolio
• Performed characterization of 5000 most interesting for
pharmacology genes – multiple patents
• Current business model - Drug Discovery
(Lexicon Pharmaceuticals, Inc.)
Nature Drug Discovery, 2003
Nature Drug Discovery, 2003
Application of transgenic models in
experimental medicine and pharmacology
• Novel target validation by screening for relevant
phenotypes of KO mice
• Testing of hypotheses of disease pathology and
uncovering novel molecular pathological mechanisms
and targets for pharmacological correction
• Recapitulation of human pathology and mutations to
develop naturalistic models of a disease
• Validation of the selectivity of action of novel
compounds
Neuronal architecture of prefrontal cortex
DA
GLU
DA
GABA+
CR-/PV-
5HT
GLU
5HT
GABA+
PV+
P
5HT
GLU
Schizophrenia susceptibility genes
are involved in the dynamic
operations of cortical circuitry
long implicated in schizophrenia,
particularly the interplay of
dopamine, glutamate and GABA .
DA
Modified from: S. Sesack and D. Lewis
Major neurochemical theories of
Schizophrenia
• Dopamine theory (enhanced activity)
• Glutamate theory (reduced activity)
Cocaine
DA
DAT
Amphetamine
DA
VMAT2
H+
AMPH
MAO
DAT-knockout (DAT-KO) Mice
Normal
Neurotransmission
Lack of
Dopamine Transporter
DAT
DA
DA
TH
TH
Extracellular DA elevated 5-fold
DA storage reduced 20-fold
DAT-KO Mouse Phenotype
Hyperactivity of DAT-KO mice
Compounds effective in inhibition of
hyperactivity in DAT-KO mice
• α-methyl-p-tyrosine, Inhibitor of tyrosine hydroxylase
• Classical (haloperidol) and atypical (olanzapine, clozapine and
aripiprazole) antipsychotics
• Psychostimulants
• Direct and indirect 5-HT agonists
• 5-HT2A antagonist M100907
• Positive modulators of AMPA glutamate receptors (Ampakines)
• Cannabinoid (CB1) receptor agonists
• Acetylcholine esterase (ACHE) inhibitors
• Nicotine
• ERK inhibitors
• Antimaniac drugs Lithium and Valproate (GSK3 inhibitors)
DAT-KO mice were licensed:
Johnson and Johnson
Servier
Cortex Pharmaceutical
Relevance of DAT-KO mice for
Schizophrenia
• Recapitulate some specific behaviors
consistent with hyperdopaminergia and
believed to be related to psychosis :
hyperactivity, persistent stereotypies,
deficient PPI, LI
• However, like amphetamine rodent models,
they do not have “classical” deficits in social
interactions, thus model is limited to
endophenotypes of “positive” symptoms
Glutamate hypothesis
• Phencyclidine (PCP) and ketamine dissociative anesthetic:
– Auditory hallucinations
– Depersonalization
– Delusions
– Both positive and negative
symptoms
• In experimental animals can be modeled by NMDA antagonist
(MK-801, PCP) - induced hyperactivity, stereotypy, disrupted
PPI and deficits in social interaction
NR1-knockdown (NR1-KD) mice (have 5% of NR1):
-Hyperactivity and stereotypy
-PPI deficit
-Hyperactivity and PPI deficits can be blocked by
haloperidol, clozapine, olanzapine, risperidone and
aripiprazole
NR1-KD mice show remarkable deficits in
social interaction reversible by clozapine
Cognitive deficits in NR1-KD mice
8-arm maze (test of spatial learning and memory)
NR1-KD mice were licensed:
• Merck
• Astellas
• Servier
• AstraZeneca
Comparison of pharmacological and genetic animal
models of hyperdopaminergia and NMDA deficiency
• Both DAT-KO and NR1 deficient mice show deficits in cognitive tasks
G Protein-Coupled Receptors (GPCR)
Seven-Transmembrane Receptors (7-TMR)
Ligands
neurotransmitters
hormones
peptides
glycoproteins
lipids
out
nucleotides
ions
tastants
photons of light
odorants
Effectors
NH2
Enzymes
Plasma
membrane
Receptor
AC, PLC
Channels
K+, Ca++
G protein subunits:
16 alpha
5 beta
12 gamma
in
COOH
G protein
• Around 1000 GPCRs are known
• 400 somatic GPCRs , 150 orphan GPCRs
• 30-40% of current pharmacology
based on targeting GPCRs
2nd messenger
molecules
cellular
response
Robert J Lefkowitz
Nobel Prize
in Chemistry 2012
b g
a
GDP
b g
GTP
a
b g
P
GRK
Effectors
Adenylyl cyclase
Phospholipase C-b
p115 RhoGEF
X
aq*
X
Effectors
P
bArr
Effectors
GPCR regulation
Clathrin
P
bArr AP2
P
Gainetdinov et al., Annu Rev Neurosci 2004
Marc Caron
Raul Gainetdinov
Bob Lefkowitz
Laura Bohn
Fang Lin
Application of transgenic models in
experimental medicine and pharmacology
• Novel target validation by screening for relevant
phenotypes of KO mice
• Testing of hypotheses of disease pathology and
uncovering novel molecular pathological mechanisms
and targets for pharmacological correction
• Recapitulation of human mutations and pathology to
develop naturalistic models of a disease
• Validation of the selectivity of action of novel
compounds
Tryptophan
Tryptophan hydroxylase (TPH)
5-HTP
Serotonin
Science 299, 76 (2003)
• Human TPH1 and TPH2 share 72% amino acid identity
• TPH2 (Chromosome 12q21.1) vs. TPH1 (Chromosome 11p14-15.3 )
BALB/CJ and DBA mice have single nucleotide mutation in TPH2
that reduces serotonin synthesis by 40-50%
Loss-of-function Mutations in Tryptophan Hydroxylase-2
(TPH2)
Tryptophan
TPH1
5HTP
Serotonin
Tryptophan
TPH2
5HTP
Serotonin
Biochem. Pharmacol. 66, 1673-1680 (2003)
TpH2 mutation in Humans
Expressed in PC12 cells
TPH2 mutation in mice
Reduced serotonin in TPH2 R439H knock-in (KI) mice
TPH2-KI mice as a model of depression:
• Impaired in deprerssion-related tests
• Display anxiety
• Aggressive
• Low CSF 5-HIAA levels
• Blunted fenfluramine-induced prolactin
response
• Blunted 5-HT(1A) agonist-induced
hypothermia
Treatment with serotonin precursor 5-HTP prevents
depletion of serotonin by SSRI in TPH2 deficient mice
Patents on TPH2 mutations
• Method for augmenting the effects of selective serotonin
reuptake inhibitors. US Patent No. 7,517,908, issued on
April 14, 2009 (Krishnan KRR, Caron MG, Zhang X,
Beaulieu JM, Gainetdinov RR, Sotnikova TD).
• Functional single nucleotide polymorphism in brainspecific tryptophan hydroxylase (Tph2) as a tool for
diagnosis and treatment of neuropsychiatric disorders.
US Patent No. 7,585,627, issued on September 8, 2009
(with Caron MG, Zhang X, Beaulieu JM, Gainetdinov
RR, Sotnikova TD, Krishnan RR, Schwartz DA, Burch
LA, Williams RB).
Novel model of acute DA deficiency,
DA-deficient DAT-KO mice
(DDD mice)
Parkinson’s Disease
Parkinson’s Disease (PD) is characterized by progressive
degeneration of dopaminergic neurons in the basal ganglia and
presence of cytoplasmic inclusions known as Lewy bodies.
Clinically manifests when loss of dopaminergic neurons reaches
around 60-70%
Major manifestations of PD :
• Resting tremor (tremor at resting conditions)
• Rigidity (tonically increased muscle tone)
• Bradykinesia / akinesia (slowness/absense of movement)
• Gait disturbance and postural instability
Other motor manifestations:
• Facial masking
• Decreased eye-blinking
1957:
Carlsson et al., Nature
• Reversal of Reserpine's
effects by L-DOPA. (Top)
Rabbits treated with
reserpine (5 mg/kg
intravenously). (Bottom)
The same rabbits 15 min
after L-DOPA (200 mg/kg
intravenously).
2000:
Arvid Carlsson won Nobel Prize
Oleh Hornykiewicz
Found that dopamine was depleted
in striatum of PD patients
First use of L-DOPA in PD patients
Novel model of acute DA deficiency,
DA-deficient DAT-KO mice (DDD mice)
Normal
Neurotransmission
(WT mice)
Lack of
Dopamine Transporter
(DAT-KO mice)
DAT
DA
DA
TH
TH
What if TH is inhibited?
DA-deficient DAT-KO mice
(DDD mice)
L-DOPA restores locomotion in DDD mice
Non-selective D1/D2 DA agonists are also effective but not as much as L-DOPA
DDD mice summary
• Maximally possible degree of acute DA
deficiency
• Remarkable motor abnormalities – akinesia,
rigidity, but normal righting reflex
• Simple screening model for drugs that can
restore movement control in conditions of severe
DA deficiency
• More than 100 compounds have been tested to
date in DDD mice
Application of transgenic models in
experimental medicine and pharmacology
• Novel target validation by screening for relevant
phenotypes of KO mice
• Testing of hypotheses of disease pathology and
uncovering novel molecular pathological mechanisms
and targets for pharmacological correction
• Recapitulation of human pathology and mutations to
develop naturalistic models of a disease
• Validation of the selectivity of action of novel
compounds
Trace Amines (TAs) and their
receptors (TAARs)
• TAs (tyramine and octopamine) are involved in movement control
and other functions in invertebrates, but role in mammals is unclear
• Can act as “false” neurotransmitters via interaction with monoamine
transporters; β-PEA interacts with dopamine transporter (DAT) like
amphetamine, inducing hyperlocomotion and stereotypies
• TAs concentrations controlled by L-AADC and MAO
•
β-PEA plasma levels are altered in Parkinson’s disease, depression
and during maniac episodes
Dopamine
β-Phenylethylamine
Amphetamine
Alpha-Methyl-PHenylEThylAMINE
Trace amines: identification of a family
of mammalian G protein-coupled
receptors. Borowsky et al, 2001
• 9-21 GPCR genes from human to
rodents were identified
•
All TAARs shares a common motifs
•
All TAARs are located in a single
chromosomes
•
All TAARs are encoded by a single
exons, except TAAR2
Phylogenetic organization of TAARs
Validation of selectivity of newly developed TAAR1 agonist
(in collaboration with F. Hoffmann-La Roche Ltd )
• TAAR1 agonist inhibits hyperactive DAT-KO mice
• This effect is absent in double KO mice lacking
DAT and TAAR1
Knockout Rats
• Better model than mice for human cardiovascular
disease, diabetes, arthritis, and many autoimmune,
neuropsychiatric and addiction disorders, cognitive
studies; better object for surgery
• It was not technically possible until 2009
• NIH established Knock Out Rat Consortium (KORC) in
2008
• SAGE laboratories developed zink-finger nuclease (ZFN)
technology and commercialized via SIGMA KO rat
development (90,000 USD per strain)
• Up to date about 100 KO rats were developed
Трансляционная биомедицина в
СПбГУ
• В Декабре 2014 г. СПБУ выиграл конкурс на
получение грантов по приоритетному
направлению деятельности РНФ «Реализация
комплексных научных программ организаций»
• Руководитель – Ректор СПБГУ Николай
Михайлович Кропачев
• Финансирование РНФ – 750 миллионов рублей
(2014-2018 г.)
Трансляционная биомедицина в СПбГУ
Чернов Ю.О.
Певзнер
П.А.
Биобанк
Алгоритмическая
биоинформатика
Гайнетдинов
Р.Р.
Красавин
М.Ю.
Тенникова
Т.Б.
Трансгенные
модели
Разработка
лекарств
Нанодоставка
лекарств
Биологический факультет
Медицинский факультет
Факультет стоматологии
Кафедра физической культуры и
спорта
Факультет психологии
Институт Химии
Клиника
Пациенты
Трансляционная биомедицина в
СПбГУ
Научная инфраструктурa:
Дополнительно к уже существующим Центрам
Коллективного Пользования будут созданы:
• 1. Криохранилище для хранения образцов
Биобанка c coответствующим современным
оборудованием
• 2. Современный вивариум для содержания
трансгенных животных удовлетворяющий
международным требованиям SPF (specific
pathogen free).
Лаборатория Трансляционной Нейронауки и
Молекулярной Фармакологии
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Татьяна Сотникова, с.н.с. СПБГУ, Researcher, Italian Institute of Technology,
Genova, Italy
Евгений Будыгин, с.н.с. СПБГУ, Assistant Professor, Wake Forest University,
Winston Salem, NC, USA
Евгения Ефимова, н.с., СПБГУ, постдок, Сколтех
Андрей Герасимов, постдок, СПБГУ
Евгений Аккуратов, постдок, СПБГУ
Евгений Благовещенский, н.с. СПБГУ
Наталья Аккуратова, аспирант, Сколтех
Наталья Католикова, аспирант, СПБГУ, Сколтех
Мария Михайлова, аспирант, СПБГУ
Работа Лаборатории финансируется:
• Грант СПБГУ для научных групп, №1.38.201.2014 (2014-16)
• Грант РНФ для научных групп, №14-15-00131 (2014-16), руководитель Сотникова Т.Д.
• Грант РНФ для существующих лабораторий и кафедр, №14-25-00065 (Кафедра Высшей
Нервной Деятельности, Биологический Факультет СПБГУ, 2014-2016)
• Грант РНФ для реализации научных программ организаций, № 4-50-00069 (2015-2018)
• Сколтех