WO1996035785A9

WO1996035785A9 - Transgenic animals having a defective thyroid hormone receptor beta gene

Info

Publication number: WO1996035785A9
Application number: PCT/EP1996/001983
Authority: WO
Filing date: 1996-05-10
Publication date: 1997-02-06

Abstract

The invention provides a transgenic mammal which is heterozygous or homozygous for an at least partially defective thyroid hormone receptor β gene, cells derived from the mammal and methods for the use of the mammal and the cells.

Description

TRANSGENIC ANIMALS HAVING A DEFECTIVE THYROID HORMONE RECEPTOR BETA GENE

This application relates to transgenic animals, particularly mice, and tissues and cell lines

thereof that in a homozygous form lack the gene for thyroid hormone receptor β (TRβ). The

mice, tissues and cell lines of the invention may be used in the testing for pharmaceutical or

clinical purposes of substances such as thyroid hormones T₃ and T₄ and possible antagonists

and agonists thereof.

The thyroid hormones tri-iodothyronine (T₃) and thyroxine (T₄) have a very wide range of

effects. Li adult mammals they influence nearly all organs, the metabolism of nutrients, basal metabolic rate and oxygen consumption. In humans, the deficiency or excess of circulating thyroid hormones results in the well characterised syndromes hypo- and hyperthyroidism.

The thyroid hormones are essential for the normal development of the central nervous system

particularly in the foetal and neonatal stages ^1"6. Deficiencies in the action of thyroid

hormones lead to hypothyroidism that can be due to either acquired or congenital disorders.

Some of the congenital causes of hypothyroidism are embryopathies as absence, hypoplasia, or ectopic localization of the thyroid gland; enzymatic disorders; deficient hormone synthesis and receptor disorders (Generalized Thyroid Hormone Syndrome (GRTS)). Unless treated, congenital hypothyroidism leads to irreversible mental retardation and short stature

(dwarfism). Other symptoms include neurological dysfunctions such as poor coordination and

balance, abnormal fine motor movements, speech problems, spasticity, tremor and hyperactive

1 deep tendon reflexes. In addition basal metabolic rate, gluconeogenesis, lipogenesis and cardiac output are decreased. Hypothyroidism in adults leads to symptoms similar to those

described above, except for the mental retardation. However, adult patients are easily treated

with hormone therapy.

In contrast to congenital hypothyroidism, hyperthyroidism is more common in adults. In general, the symptoms are the reverse: increased metabolism, lower serum cholesterol levels,

hyperactivity and tachycardia are hallmarks of elevated T3/T4 levels ²⁵.

Thyroid hormones act through thyroid hormone receptors (TRs) which belong to the

superfamily of steroid hormone receptors. TRs are ligand dependent transcription factors which regulate the transcription of their target genes through responsive elements in the DNA. In vertebrates there are a variety of TRs^7"13 (Fig. A) derived from TRα and TRβ genes, which

are located at the 17th and 3rd chromosomes respectively in humans. There is considerable

homology between the TRα and TRβ proteins and between the receptors in different species, such as rat, mouse, and human. The α-gene encodes the subtypes l and α2. The α2

subtype is not a functional receptor in the sense that it lacks T -r₄ hormone binding capability.

The β-gene encodes the subtypes β 1 and β2. The latter has so far been identified only at the

messenger RNA level. The physiological significance of these different proteins has not yet

been clarified. Different amino- and carboxy-termini for the TR variants suggest different trans-activating properties for TRα and Trβ. In addition, the differential expression during brain development suggest different roles for the TR variants during development ^14"16. The mechanism of T₃ action via its receptor is quite complex due to the presence of multiple TRs ^17"20. The TRα locus encodes in addition to the TRα gene another receptor denoted as

Rev-α. Rev-α arises by transcription of the opposite strand of TRα gene and overlaps the α2

region at the 3' end (Fig. B). Furthermore, there are TRα2 and TR 3 variants; the protein

sequence of the latter is identical to that of TRα 2 with the exception that it lacks the first 42

amino acids of the carboxy terminus (Fig. C).

In humans, the GRTS has been related to TRβ gene disorders. No clinical syndromes have

yet been associated to TRα gene mutations suggesting that the TRα gene is either dispensable

or essential for life. It is equally unclear as to which of the two thyroid hormone receptors the actions of thyroid hormones can be ascribed in hypo- and hyperthyroidism. If the individual

functions in hormone action of the receptors could be identified, agonists or agonists that are

specific for either ofthe receptors could be used for treatment of specific target tissues without

adversely affecting other tissues.

Treatment of many diseases associated with thyroid hormone function cannot be done today

since administration of increased doses of the hormone to achieve a desired effect in a given tissue, leads to adverse effects in another. The effects of thyroid hormones are mediated by

two different receptors that are coexpressed in some tissues, whereas other tissues express only one of them. It should therefore be possible to design agonists and antagonists that are specific for each of the receptors and that can mediate a desired activation or repression of

receptor function. In order to allow testing of such components we have disrupted the TRβ gene in the mouse genome, and bred such animals to homozygosity. These animals can grow to at least sexual

maturity, and are therefore suitable tools for identifying the action of agonists and antagonists

of TRβ.

According to one aspect of the invention there is provided a transgenic mammal which is

heterozygous for an at least partially defective thyroid hormone receptor β gene. The

defective gene may be inactivated for example by an insertion, deletion, substitution or inversion or any other suitable genetic manipulation.

Preferably, the mammal is a rodent, more preferably a mouse.

One heterozygous transgenic mammal in accordance with the invention may be bred with another such heterozygous transgenic mammal to produce a mammal which is homozygous for a defective thyroid hormone receptor β gene. Thus according to another aspect of the

invention there is provided a transgenic mammal which is homozygous for an at least partially

defective β thyroid hormone receptor β gene.

The invention also provides cells derived from the animal of the invention which are heterozygous or homozygous for a defective thyroid hormone receptor β .

According to another aspect of the invention there is provided a method of producing a

transgenic animal in accordance with the invention the method comprising : 1) preparing a gene encoding an at least partially defective thyroid hormone receptor β as described above;

2) introducing that gene into suitable carrier cells;

3) inserting those carrier cells into an embryo; and

4) replacing the embryo into a mother, and allowing the embryo to develop to full term.

According to a further aspect of the invention there is provided a method of testing the

agonist/antagonist properties of a compound in relation to the thyroid hormone receptor, the

method comprising:

contacting a transgenic animal in accordance with the invention with the compound and monitoring subsequent development of the animal.

Alternatively, the method may involve using cells or tissues derived from the transgenic

animal.

The transgenic mammal of the invention is suitable for testing the effects of agonists and

antagonists of thyroid hormone action, in particular those that discriminate between TRα and TRβ. In particular, the transgenic mammal of the invention or cells or tissues derived therefrom can be used to study the following:

1. Administration of excess thyroid hormones decreases high serum cholesterol levels.

However, an adverse side effect is that cardiac output also increases which can lead to arrythmia. If these two functions of thyroid hormones are mediated by distinct receptors, a proper administration of receptor specific agonists or antagonists would lead to the desired decrease in serum cholesterol while leaving cardiac function

normal.

2. Hypo- and hyperthyroidism adversely affect bone structure. The use of receptor-

specific thyroid hormone antagonists or agonists for treatment of e.g

hypercholesterolemia or other diseases must therefore include a test for their influence on bone synthesis and turnover.

3. Regulation of heart functions such as pulse, arrythmia, or myocardiac muscle can be

targeted by the use of receptor specific thyroid hormone antagonists or agonists.

4. Many organs or tissues produce hormones in a thyroid hormone dependent manner. Such tissues include the hypophysis (producing growth hormone, prolactin, thyroid stimulating hormone, luteinizing hormone), the hypothalamus (thyrotropin releasing hormone, oxytocin), peripheral tissues (insulin growth factor I). The effect of receptor

specific thyroid hormone antagonists or agonists on such endocrine systems can be

determined with the mammals of the present invention.

5. Basal metabolic rate, gluconeogenesis, lipogenesis, lipolysis and thermogenesis are

increased in hyperthyroidism and decreased during hypothyroidism. The effect of

receptor specific thyroid hormone antagonists or agonists on such metabolic processes

can be determined with the mammal of the present invention. 6. Toxic effects of agonists and antagonists on normal and abnormal physiological metabolic processes.

7. Effects on brain or other neuronal function (hearing, peripheral nervous system), as

well as effects on embryonal and foetal development of receptor specific thyroid

hormone antagonists or agonists on such endocrine systems can be determined with

the transgenic mammal of the present invention.

8. Effects on increasing or decreasing body growth in patients with growth disorders.

9. A large number of genes or gene products are known to be regulated by thyroid hormones. The effects of agonists and antagonists of such systems before clinical

trials can commence.

10. Effect on haemopoiesis. Hypothyroid patients are usually anaemic.

11. Treatment of patients that have defective TRα receptor genes. As mentioned above,

no patients with mutant TRα genes have been found, whereas genetic defects in more

than 250 patients with defective TRβ genes have been identified. The latter patients were first clinically identified due to their inappropriate levels of thyroid hormones and other thyroid hormone regulated hormones such as TSH. It is therefore possible

that diseases due to defects in the TRα gene have remained undetected because the

patients have normal T₃/T₄ and TSH levels and their symptoms therefore would not be easily associated with a receptor dysfunction. The TRβ deficient mammals of the present invention allow the identification of such disease, symptoms, and their cure

with suitable agonists.

Mammals in accordance with the invention and their production will now be described by way

of example only with reference to the further accompanying drawings Figures 1-2 in which:

Fig. 1 illustrates disruption of the TRβ gene by homologous recombination; and

Fig. 2 illustrates an RT-PCR analysis of products of the wild type and mutant alleles of the

TRβ gene.

Example 1

Generation of mutant mouse with deleted thyroid hormone receptor β gene.

EXPERIMENTAL PROCEDURES

Targeting vector

A chick TRβ cDNA insert was used to screen a bacteriophage lambda library of genomic

DNA of a 129sv strain mouse (Stratagene) to obtain overlapping clones that encompassed the entire coding domain of the TRβ gene. Fig. 1 A is a schematic representation of the TRβ 1

protein showing the central DNA binding domain (filled in black) and C-terminal T3-binding

domain. Fig. IB top line, illustrates the structure of the central region of the gene containing

the first three coding exons that are common for both TRβ 1 and TRβ 2 (here numbered 3 to 5). The middle line illustrates the targeting vector contained 3 kbp and 4 kbp respectively of

5' and 3' homologous flanking DNA and canied a 3 kbp deletion including part of exon

number 3. The bottom line shows the structure of the mutant allele generated by homologous

recombination 5', neo and 3' probes used in Southern blot analyses are shown as well as the

band sizes predicted to be detected with the 3' probe following digestion with Barn HI and

Eag I: the wild type band size being 19 kbp whereas the mutant band is 10 kbp. Restriction

enzyme sites are indicated where relevant. X, Xba I; B, Bam Ht; K, Kpn I; E, Eag I. The exon

structure was confirmed by DNA sequencing of plasmid sub-clones. The targeting vector

(Figure IB) contained from 5' to 3': a TK gene fragment from pMCI-HSV TK, a 3 kbp

fragment of TRβ genomic DNA extending to a Kpn 1 site in the coding exon number 3, a

neomycin resistance gene from pgkneobpA, a 4 kbp Xba-I-Hind HI genomic fragment containing the TRβ exons 4 and 5. The construct was linearized at the 5 ' end of the TK gene

by Bam HI digestion prior to electroporation.

Electroporation and selection of ES cells

W9.5 male ES cells derived from 129/sv mice were grown on feeder layers of G418-resistant

primary mouse embryo fibroblasts (PMEFs) in dishes that had been treated with 0.1 % gelatin:

PMEFs were mitotically inactivated by gamma-hradiation. W9.5 cells were cultured in Dulbecco's Modified Eagle medium (Specialty Media) supplemented with 15% defined fetal bovine serum (Hyclone), 1000 U/ml of recombinant LIF (Gibco), L-glutamine, non-essential amino acids, β-mercaptoethanol and antibodies as described ²⁶ 3 x 10⁷ W9.5 cells at passage

12 were resuspended in 0.8 ml PBS containing 25μg of linearized targeting vector DNA for

electroporation using a Bio-Rad Gene Pulser (500μF, 250V). Cells were then plated onto 60mm dishes. The next day the medium was replaced with medium containing 350 μg/ml G418 (dry weight, Gibco) and on day two, 2μM ganciclovir (a gift of Syntex Corp. Palo Alto,

CA) was added. The medium was replaced each day and on day 8, colonies were picked and

transferred into 48 well dishes. After 4-5 days growth in 48-well plates, each clone was

trypsinized and 9/10 of the suspension volume removed for DNA preparation. To the

remaining volume, fresh medium and PMEFs were added. Clones identified as positive for

homologous recombination were expanded and stocks frozen. The chromosome content of

positive clones was determined by growth on microscope chamber slides for analysis in situ.

Southern blot hybridization analysis of ES cell clones and genotype determination

ES cells colonies were screened for homologous recombinants in pools of six. Cell pellets were lysed at 55°C overnight and DNA was prepared and digested overnight with Bam HI and Eag I, then analyzed on 0.7% agarose gels. DNA was transferred to Duralose-UV membrane and hybridized using Quickhyb solution (Stratagene) with the indicated 3' probe (Figure 1).

Membranes were washed in O.lxSSC, 0.2% SDS at 62°C twice, then once at 65°C. DNA

samples from mice were prepared from tail clips and genotypes routinely determined by digestion of 5-10 μg of DNA with Bam HI and Eag I and analysis by hybridization as described above.

Blastocyst injection and mice breeding

ES cells of recombinant clones were injected into C57B1/6J blastocysts which were then

transfeπed into pseudopregnant recipient female mice of strain C57BIJ6J. Male chimaeric offspring were obtained with extensive ES cell contribution as judged by their agouti coat colour. Five of these were bred with C57B1/6J female mice and produced agouti -coloured offspring indicating germline transmission. The genotype of these Fl mice was determined

and TRB heterozygotes were crossed to generate litters containing homozygous mutants. All

analyses were performed with progeny obtained from crosses between these TRB

heterozygotes and thus represented hybrid mice derived from 129/sv (ES cell) and C57bl/6J

strains.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of mutant gene products

Total cellular RNA from selected tissues of wild type, heterozygous and homozygous mutant

TRB mice was prepared and used to make first strand cDNA using as primer an antisense oligonucleotide derived from the 3' terminal coding exon of the mouse TRβ gene. RT-PCR analysis was then performed on the cDNA using the pairs of primers indicated in Figure 2 that

specifically amplify products representing the N-terminal coding regions of the two TRβ N-

terminal variant proteins (TRβ 1 and TRβ 2) that are encoded by the TRβ gene. The products

from mice of all three genotypes were purified and their DNA sequences were determined by

automated sequencer.

RT-PCR analysis of products of the wild type and mutant alleles

RT-PCR products of RNA from different tissues from wild type (+/+), heterozygous (-/+) and homozygous mutant (-/-) mice were generated using pairs of primers that specifically amplify products derived from TRβ 1 and TRβ2, as indicated in the lower part of the figure. Products

were electrophoresed on 0.8% agarose gels and visualised by ethidium bromide staining. In

all tissues from homozygous mutant mice, the RT-PCR products were 100 bp shorter than in NOT TO BE TAKEN INTO CONSIDERATION FOR THE PURPOSES OF INTERNATIONAL PROCESSING

Example 2

Analysis of the effect of the thyroid hormone receptor β on the development of auditory

function

Mice which were heterozygous (Thrb^"/+) were prepared as described above. The auditory-

evoked brainstem response (ABR) was tested in these mice. It was found that the threshold

sound pressure levels required for ABR were significantly elevated (p«0.01) for all pure

tones tested (8,16 and 32 kHz) and for a click stimulus in all adult Thrb^"'^" mice. Thrb^"/+ and

control Thrb^{+ +} mice both had ABR thresholds in the normal range, whereas Thrb^"7" mice

displayed significantly elevated thresholds that were often in the 70-100 dB range, indicating severe impairment. Indeed, 10-15% of Thrb^"'^" mice were profoundly deaf since no response could be evoked with any frequency tested at 100 dB, the upper limit of the apparatus. In

mutants in which a response could be evoked, albeit with elevated thresholds, the resultant

ABR waveforms were not significantly different from those ofthe controls, with normal peaks

and latencies, indicating that brainstem auditory functions were normal and suggesting a defect in the generation of the primary action potential from the cochlea. Since the impairment was general with respect to all frequencies tested, the defect was not restricted to particular regions of the cochlea that are responsive to specific frequencies. There was not evidence for vestibular defects, since Thrb^"'^" mice showed no circling or other abnormal behaviour. Analysis of mice at 2-3 weeks of age when hearing normally approaches adult sensitivity levels, also demonstrated impairment in Thrb^"'^" mice (p«0.01) compared to

controls. This confirmed that the mutation caused a permanent failure of development of

auditory function. Example 3

Physiological effects of targeted interaction of the mouse Trβ gene.

Thyroid pathology in homozygous mutants

Thrb^"'^" mice produced as described above were viable, they displayed normal growth rates and

weight gain and they were fertile. Necropsy failed to reveal gross abnormalities in most

organs, with the exception of the thyroid gland which was variably enlarged in Thrb^"'^" mice.

Quantitative image analysis of histological sections indicated that thyroid areas were 1.5-2.0

fold increased (P<0.05) in overall size in homozygotes (mean ± SEM in mm², 0.58±0.09, n

= 10) compared to heterozygous (0.35±0.04, n = 9) and wild type (0.39±0.04, n = 8) mice at 5 weeks of age. There was no significant difference between Thrb ⁺ and Thrb^{+ +} mice. Higher

magnificent revealed a diffuse enlargement of Thrb^"'^" thyroid glands resulting from an increase

in both the numbers and size of follicles. The colloid of follicles from Thrb^"'^" mice frequently contained large phagocytic-like cells that were often multi-nucleated and other cellular debris that was probably derived from degenerating epithelial cells.

This pathology suggested that the Thrb^"'^" thyroid glands were in a hyperactive state with

increased epithelial cell turnover, indicating that the mutation caused a recessive hyperthyroid- like condition. No difference was detected between the sexes and the enlargement persisted

in mice analysed at 5, 18 and 40 weeks of age. The condition was not progressive since the pathology was not more pronounced, with no evidence of hyperplasia, in 40 week old mice.

Image analysis of thyroid sections demonstrated an approximately constant ratio of areas of

colloid:epithelium in Thrb^"'^" (mean ± SEM, 1.02±0.08, n - 10). Thrb^"'⁺ (0.86±0.1, n = 9) and Thrb^{+ +} (0.91±0.1, n - 8) mice. Thyroid size increased in all genotypes with age, but there was

no significant difference in the ratio of colloid:epithelium between Thrb^"'^" and normal mice. The thyroid glands of Thrb^"'^" mice at postnatal day 7 also displayed an increase in the numbers and size of colloid-containing follicles indicating that the condition arose at an early age.

Hormonal disorder

The observed thyroid pathology ofthe Thrb^"'^" mice suggested that there could be abnormalities

in thyroid hormone levels. Analysis of serum thyroid hormones revealed that the levels of

total thyroxine (TT4), the major product of the thyroid gland, were significantly elevated in

Thrb^"'^" mice at 5 - 40 weeks of age, inespective of gender. Fig. 4A shows that mean TT4 levels were elevated -2.5 fold in a representative analysis of 10 week old mice (means ±SEM

for Thrb^"'^", Thrb^'/+ were 11.5±1.07, 4.6±0.3, 4.1±0.3 μg/dL, respectively). Parallel increases

in free T4 were observed in Thrb^"'^" mice (1.7±0.18 ng/dL) compared to Thrb^{' +} (0.6±0.05) and

Thrb ^{+ +} (0.5±0.06) mice. This confirmed the predicted thyroid hyperactivity and excluded abnormal serum binding or transport of T4 as the cause ofthe elevated serum hormone levels.

Preliminary data indicated that there was a general decrease of TT4 levels in older Thrb^"'^" mice (-1.5 years of age), suggesting that the hyperactivity was ameliorated with age. The levels of total and free T3, the main biologically active form of thyroid hormone, were also elevated in Thrb^"'^" mice. The levels of total T3 were somewhat variable regardless of the genotype, perhaps indicating variability in the peripheral conversion of T4 to T3 in this mouse strain.

However, free T3 levels were consistently elevated.

Failure to regulate thyroid stimulating hormone Elevation of thyroid hormone levels normally suppresses TSH production by the pituitary thyrotropes. However, the mean serum levels of TSH were significantly elevated in Thrb^"'^"

compared to Thrb^"'⁺ or Thrb^+/+ mice at 5-40 weeks of age, inespective of gender. Thus,

despite the high levels of thyroid hormones, TSH was paradoxically elevated in Thrb^"'^"

mutants. Northern blot analysis of pituitary RNA showed that levels of mRNA encoding

TSHα and TSHβ subunits were elevated 2.5 and 3.3-fold respectively compared to Thrb^+/+

mice, suggesting that the increased TSH levels in mice lacking Trβ reflected abnormal

regulation of TSH gene transcription. Histological examination of pituitary glands from

Thrb^''^" mice revealed no abnormalities and immunohistochemical analysis showed no

abnormal pattern of cells straining positively for the TSH subunits (Fig. 4D-G). Thus, the

over-production of TSH detected in Thrb^"'^" mice resulted from defective thyrotrope function

rather than from hypeφlasia malformation of the pituitary gland.

Central nervous system (CNS) function and anatomy

The absence of, or excessive exposure to T3 during a critical embryonic and neonatal period can impair brain development (Legrand, 1984). To investigate if the absence of Trβ and/or

the associated increase in thyroid hormone levels caused neurological defects, the function of

the nervous system in Thrb^"'^" mice were assessed using a range of behavioural tests. These analyses were valid since mice, like humans or rats, are susceptible to behavioural defects associated with congenital thyroid disorders and similar tests have demonstrated learning

disabilities in the hypothyroid (hyt) mutant mouse (Anthony et al., 1993). In a stringent

version of the Moπis water task, requiring the mice to locate a hidden platform to escape.

Thrb^"'^" and Thrb^{+ +} mice learned to escape equally well with repeated trials over nine days. When the platform was removed, mice of both genotypes spent equivalent time and activity

in the quadrant where the platform had been located. Context fear conditioning and responses

to paired stimuli that may indicate attention deficits were not significantly different in Thrb^"'^"

mice (data not shown). However, these studies may not be conclusive as they employ an

acoustic stimulus to which the mutants could not respond reliably due to defective auditory

function (Foπest et al, submitted). In other tests such as activity in an open field and Y-maze,

Thrb^"'^" and Thrb^{+ +} mice also behaved similarly. Histological and histochemical analysis of

the CNS of Thrb^"'^" mice revealed no obvious abnormalities in brain anatomy, including

structures known to be sensitive to T3, such as the cerebellum hippocampus. Furthermore,

analysis of hippocampal field potentials did not indicate defects in long term potentiation. In conclusion, while development delays and attention deficits were not excluded, no overt neurological defects were detected in adult Thrb^"'^" mutants, suggesting that Trβ has subtle

rather than major functions in neurodevelopment.

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Claims

CLAIMS:

1. A transgenic mammal which is heterozygous or homozygous for an at least partially defective throid hormone receptor β gene.

2. A transgenic animal according to claim 1 in which the defective thyroid hormone

receptor β gene has been produced by an insertion, deletion, substitution or inversion

or other suitable genetic manipulation.

3. A transgenic animal according to claim 1 which is a rodent.

4. A transgenic animal according to claim 3 which is a mouse.

5. Cells derived from the transgenic mammal of claim 1 which are heterozygous or homozygous for defective throid hormone receptor β.

6. A method of producing a transgenic animal in accordance with claim 1 , the method

comprising the steps of:

1) preparing a gene encoding an at least partially defective thyroid hormone receptor β as described above;

2) introducing that thyroid hormone receptor β gene into suitable carrier cells;

3) inserting those carrier cells into an embryo; and 4) replacing the embryo into a mother, and allowing the embryo to develop to full term.

7. A method of testing the agonist/antagonist properties of a compound in relation to a thyroid hormone receptor, the method comprising the steps of:

contacting a transgenic animal in accordance with claim 1 with the compound and monitoring the subsequent behavioural development of the animal.

8. A method of testing the agonist/antagonist properties of a compound in relation to a thyroid hormone receptor, the method comprising the steps of: contacting cells in accordance with claim 5 with the compound and subsequently monitoring the cells.