WO2001038535A1 - Chimeric gene enabling expression of the gene or cdna of type i insulin-like growth factor (igf-i) in the pancreas and utilization thereof in genic therapy of diabetes mellitus - Google Patents

Abstract

The invention relates to a chimeric gene using the gene or cDNA of type I insulin-like growth factor (IGF-I) directed by a promoter or promoter fusion enabling regulated expression of IGF-I in the pancreas. The invention also relates to an expression vector enabling the expression of IGF-I in the pancreas. The invention further relates to a pancreatic cell expressing said chimeric gene. The invention additionally relates to the utilization of said chimeric gene or said expression vector in the development of therapeutic application for diabetes mellitus.

Description

CHEMICAL GENE THAT ALLOWS TO EXPRESS THE GENE OR CDNA OF THE SIMILAR GROWTH FACTOR TO TYPE I INSULIN (IGF-1) IN PANCREAS AND ITS USE FOR MELLITUS DIABETES GENE THERAPY

FIELD OF THE INVENTION

The present invention relates to a chimeric gene containing the gene or the cDNA (DNA ^ ^ mr ^ l pτn_ i "ar ι ^ H = _. 1 fr * r- r. __ r <-v ~ __ r * i τrι "i £ __ _> r ~ ι + - ^ i η -i ^" i -__ -r- n 1 __j type I insulin (IGF-I) led by a promoter or fusion of promoters that allow to express IGF-I in pancreas and its use for diabetes m ° ilitus therapy

BACKGROUND OF THE INVENTION

Diabetes mellitus is the most common metabolic disease. It comprises a wide variety of syndromes with different etiologies that collectively affect 2 to 7% of the world population. From 5 to 10 ° OR of patients can be grouped into the category of insulin-dependent diabetes mellitus or type 1 diabetes, which usually manifests before age 40, often during adolescence, and is the result of the autoimmune destruction of the β cells of the islets of Langerhans in the pancreas, leading to insulin deficiency, hyperglycemia and the development of microvascular, acrovascular and neurological complications. The risk of these complications INCREM.ENCODER ^t according to the degree of hyperglycemia _^ ^c patients with type 1 diabetes depend dramatically on the adminis ^t r tion of insulin. The interruption or lack ^ _^ l Treatment with insulin leads to ^ lucrar - ¹ hyperglycemia, then to coma and finally to death of the patient if the hormone is not injected. While insulin therapy allows most patients to lead an active life, this substitution is imperfect and greatly affects their lifestyle. Intensive insulin therapy (three or more daily injections) can delay and slow the onset and progression of

l r-ioc: mi r n pc 'nl aroc Qi η OTTI _3 irrrry oct-a clase de tratamiento no se puede llevar a cabo en todos ]_ : ⁿ_LCiβπtss diabéticos siendo d°saconse^~^ abie tanto en

nr-ςηnsc: mp π αc 7_ H __rη á c 1 C pri αn+"Dc scieticics a esι_.e ur ι_amienι_o intensivo con insulina presentan un elevado riesgo de sufrir episodios de h- n fl ti αmi a1 _í

l r-ioc: my rn pc 'nl aroc Qi η OTTI _3 irrrry oct-a treatment class cannot be carried out at all] _: ⁿ _LCiβπtss diabetic being d ° saconse ^~ ^ open both in

nr-ςηnsc: mp π αc 7_ H __rη á c 1 C pri αn + "Dc scieticics a esι_.e ur ι_amienι_o intensive with insulin have a high risk of suffering episodes of h- n fl ti αmi a

Currently, the majority of patients are treated with subcutaneous injections of recombinant human insulin preparations that attempt to mimic the physiological profiles of the hormone (low baseline levels to which postpandial peaks of insulin secretion overlap.) The insulin administered in solution is rapidly absorbed, and is fast acting, while insulin particle suspensions of different sizes provide long and intermediate action, however, the soluble insulin mixture with slow reduces the availability of fast acting components [Heine et al. (1985 ) Bio. Med. J. 290, 204-205] One of the main deficiencies of delayed-acting insulins is variable absorption from subcutaneous tissue [Binder et al. (1984). Diabetes Care 7, 188-199 ] In addition, delayed-acting preparations are not usually capable of producing adequate basal insulin levels, resulting in many cases. you are in the appearance of both hyperglycemia and hypoglycemia. Therefore, the levels of circulating insulin that are achieved with current therapies they are quite far from those that would be obtained if the β cells functioned correctly.

Because insulin replacement therapy is not perfect, pancreas transplants and islet transplants have been attempted [Remuzzi et al. (1994). Lancet 343, 27-31]. These approaches are intended to eliminate daily insulin injections, but require chronic immunosuppression and the results have not been very successful. In addition, donors are very limited and, therefore, the treatment of a large number of patients does not seem very realistic. Another approach is based on the regeneration of the β cells from the precursors of the islet cells or on the induction of the proliferation of those β cells that have not yet been destroyed during the autoimmune process in diabetic type patients. one.

It has been observed that the expression of IGF-I increases in the areas of regeneration after partial pancreatectomy, which suggests that IGF-I may have an important role in the growth and differentiation of pancreatic tissue

[Smith et al. (1991) Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas. Froc Na zi.

Acad. Sci. U. S. A. 88, 6152-6156]. Thus, the local expression in the pancreas of insulin-like growth factor type I (IGF-I) would make it possible to counteract type 1 diabetes by inducing proliferation and neogenesis of pancreatic islets. Insulin-I-like growth factor (IGF-I is a polypeptide that has high sequence homology with insulin [Le Roith, D. (1997) Insulin-like growth factors. N. Engl. J. Med. 336, 633-640; Schofield, PN

(ed.) (1992). The insulin-like growth factors: structure and biological function. Gxford Uni v. Press, Oxford]. Its main action is to stimulate proliferation and Cell differentiation. Circulating IGF-I is produced by the liver, although it has been observed that in the adult mouse the pancreas expresses high levels of IGF-I [Mathews et al. (1986) Regulation of i.nstiii.π ~~ 1i.l Θ cro t_n f3.cto._r I ΘΠS θ sssiorj. 3o ^r _ro t__ hormone. Proc. Nati Acad. Sci. USA, 83, 9343-9347]. Our approach to chronic therapy of 13 di betes is based on the use of vectors that allow the ^" pancreas to ^r e rduducs JGF ¹ ""1^" i τ ¡= _ to be manipulated. 1 ^ rln π ir í p 1 p rc_ rfD Q rp ^ ι ήn H 1 ac. * - ά ^" I π ^" I __. c: <icas oancreá by a mechanism or au ^~ ocrino paracrinc action, to thereby recover insulin production) ar ^~ e of the pancreas of the patient.

DESCRIPTION OF THE INVENTION

The present invention relates to a chimeric gene that uses the insulin-like growth factor gene or cDNA of type I 'IGF-1 ^ directed by a promoter or fusion of promoters that allows expression of IGF-I in the pancreas during the diabetic process. This chimeric gene may contain a promoter or fusion of romotores ^p allowing ex ^p ressure regulated IGF-I in endocrine or exocrine pancreas during the diabetic process.

The present invention also relates to an expression that allows to express the chimeric gene described above in pancreas. Said vector may be a platinum, a viral vector or a non-viral vector. When it is a viral vector, it can be a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a Sindbis viral vector, a lentiviral vector or a vector derived from herpes virus.

The use of the chimeric gene for the development of approaches is an object of the present invention. -ror-aτ θi'ιτ-i -a Q for ^" I ^ Hi jp oc mol 1. nc to Q i romπ 1 p use of the expression vector of the invention for use in the development of therapeutic approaches for diabetes mellitus An in vivo approach to the gene therapy of diabetes was carried out by obtaining transgenic animals that overexpress IGF-I in pancreatic β cells under the control of the insulin gene promoter.In particular, said transgenic animal is a r ^^ ón

First, the chimeric gene was obtained and it was icroinjected into fertilized oocytes to generate the mice

transgenic, the tissue distribution of the transgene expression was determined. Thus, the presence of IGF-I mRNA in the pancreas of the transgenic mice was determined, indicating that the insulin promoter was able to direct the expression of IGF-I in those tissues where the insulin gene is expressed. endogenous These results indicated that the expression of IGF-I by the pancreas ^ __ ^ι _ - these transgenic animals were combatible

~

__H_aH ^ _ r -__- -poc: hrpnt; rτ_ τ r 'c: __] i TnDπt-pHnq αcαn phpη levels of both insulin and serum glucose ιmilar ^c ° sa those in control mice' glucose: 14,016 g / dl control versus 13715 mg / dl transgenic; insulin:

1 fl + O "^ n ^_" T / τηl í-nnl-rnl fron-Q at 1 S + Q? "-> transgenic πTTT / τr.1).

Transgenic mice that overexpress IGF-I have islet hyperplasia with age (more than 6 rnoc;. P "> ~ __ ^" 1 M "(- ι __H'i τηπS al _a c Η π H" in Hol f imn t- prni ΩΠ -tr. of such transgenic mice in inducing conditions h ^"θ ^ ^ diucem ara determine whether these animals could counteract diabetic disorders, caused by the treatment with Stz, by forming new islets or by inducing the regeneration of residual islets. For this, experimental diabetes was induced in both control mice and transgenic mice by injection for five consecutive days of the toxic streptozotocin (Stz) intraperitoneally. The administration of Stz at low doses for five consecutive days leads to the development of inflammation and autoimmune destruction of β cells

[Rossini et al. (1977). Genetic influence of the streptozotocin-induced insulitis and hyperglycemia.

Diabetes, 26, 916-920].

First, serum glucose and insulin concentrations were determined 30 days and 90 days in controls and transgenics and 180 days in transgenics after the last Stz injection as shown in. Table 1 below.

Table 1. Serum glucose and insulin levels of animals treated with STZ

day 0 day 30 day 90 day day 30 day 90 day 180 Glucose 140 + 6 579 + 16> 700 137 + 5 530 + 32 225 ± 30 215 + 23 (mg / dl) Insulin 1.5 ± 0.2 0.6 ± 0.1 0.3 ± 0.1 1.8 ± 0.3 0.7 ± 0.1 1.5 ± 0.4 1.6 ± 0.4 (ulU / l

insulina en los islotes, lo cual indica que se ha

rs H ea ronl i rari ήn S 1 p c -' ^"1 n 1 c; P Además, se observa un gran incremento en número de nuevos i q l a rír-hi - '-• ] n 1 a H ____ 1 pe

^τ / nnq disminución en la apoptosis. Estos resultados indican que la ^rar s ferer ci del ^rer del IGF—I a páncreas de n cientes de diabetes tipo 1 induciría un proceso de proliferación crue llevaría a un incremento en la masa de células β v por tanto, a una terapia curativa de esta enfermedad.Thus, it is observed that after treatment with Stz all control animals develop great hyperglyceia, since they lack insulin, and do not survive more than three months after treatment. On the other hand, transgenic animals that express IGF-I treated with Stz, after developing hyperglymia, recover normoglycemia 2 months later rio 1 + - a - ai ⁰¹ <->, mapt- i pppn αl rnrnp al; αrι beyond one year of treatment with Stz. Histological and immunohistochemical analyzes of the pancreas of these transgenic animals show that although there is a process of insulitis in the pancreatic islets in the initial stages, it disappears later,

insulin in the islets, which indicates that it has

rs H ea ronl i rari ήn S 1 pc - '^" 1 n 1 c; P In addition, there is a large increase in the number of new iqla rír-hi -' - •] n 1 a H ____ 1 pe

^τ / nnq decrease in apoptosis. These results indicate that the rar ^ s Ferer ci ^r er of IGF-I to pancreas n cients type 1 induce diabetes a process of proliferation crue lead to an increase in cell mass β v therefore a curative therapy of this disease

E-TEMPLES

The following examples are illustrative and not limiting is of the invention. Unless indicated

__rrτn 1 D3mn i arnDrHn con las instrucciones del fabricante.^ n rra ^ n ^" i _: r ^{Q -} 'tí VOS Sg θb ^_ UVÍθrθ-ld ° nr πα__H.Λrαc" r- * "i _; 1 c:' -

__rrτn 1 D3mn i arnDrHn with the manufacturer's instructions.

Example I. Obtaining a chimeric gene that includes the insulin-like growth factor cDNA of

For the construction of the chimeric RIP / IGF-I gene, the plasmid pKCR3 containing the rabbit β-globin gene was used. The BamHI-XhoI fragment of said gene is introduced: o at the Ba HI-XhoI restriction sites of the polyliner of the Bluescript SK- plasmid vector. This fragment of the β-globin gene contains the last two exons, the last intron, and the 3 ^' region linked to the SV40 enhancer. The SacI-BamHI fragment (-570 bp to +3 bp) of the rat-I insulin promoter (RIP-I) was introduced at SacI-BamHI sites in the polylinker of the Biuescript vector. Finally, the EcoRI fragment (577 bp) containing the complete cDNA of the rat IGF-I was introduced into the EcoR.1 target of the second exon of the β-globin gene. The resulting final plasmid was named

Example II Transgenic mouse expressing type I insulin-like growth factor cDNA (IGF-I) in pancreatic β cells.

II.1. Generation of the transgenic mouse

The transgenic mouse was obtained from microinjection of the 3.4 Kb Sacl-Xhol fragment containing the RIP / IGF-I chimeric gene described in the previous example. the fertilized occites were collected by emptying the oviducts of the donor mice 12 hours after mating. Next, 2 to 3 pl of a DNA solution (4 ng / μl) was injected into the zigcto pronucleus. Injected zygotes were implanted in the oviduct of pseudogestant females, approximately 10 zygotes per side [Hogan et al. (1986) Manipulating the Mouse E bryo. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York].

At 3 weeks after birth the presence of the transgene in the offspring was analyzed by Southern {Southern bl ot) transfer and hybridization analysis of 10 μg of DNA from the tail of each animal. The DNA samples were digested with a suitable restriction enzyme, resulting in the fragments istic characteristics, the presence of which only ° s detectable in transgenic mice.

II.2. Transgene Expression Analysis

5 The expression of the chimeric gene in the pancreas of the transgenic mice could be checked by analysis of the total RNA extracted from the pancreas of the control and transgenic animals. Total RNA was obtained using the isothiocyanate method and then O caaa -τm £ _-_ c_: + - vp; H ^Ω Ü Mü ¿a ri n rr ¿1 H o __. ! or t-mfp α c "iq of agarose containing for aldehyde was transferred [Northern blot, to a membrane for hybridization with an IGF-I probe. To do this, said probe was labeled with [a- 32p] clCTP following the oligcpriming method according to the manufacturer's instructions 5. Transfer membranes were contacted with Kodak XAR-5 films. The presence of hybridization in the RNA samples corresponding to the pancreas in the transgenic mice indicates the expression of the chimeric gene at 0 said tissue Subsequently, pancreas from control and transgenic animals were obtained and included in paraffin.The histological sections obtained from these pancreas were analyzed by immunohistochemical studies using a specific anti-IGF-I antibody 5. This allowed the presence of IGF protein to be detected. -I specifically in pancreatic β cells, indicating that the chimeric gene was adequately expressed.

0 II.3. Analysis of glucose and insulin levels

Blood samples were obtained from control and transgenic mice before (day 0) of streptozotocin (Stz) treatment and at (day) and (day) days after treatment. The glucose concentration was determined by the Bayer Giucometer Elite ™ system. Insulin was determined by radioimmunoassay (RIA) with the commercial INSULIN-CT kit from CIS Biointernational, France. 5

II.4. Obtaining diabetic mice

Experimental diabetes was induced in control and transgenic mice of weeks of age by intraperitoneal injection for 5 consecutive days of

10 40 mg Stz / kg live weight [Rossini et al. (1977) J. A. Diab. Assoc. 26, 916-920]. Stz was dissolved in a solution of sodium citrate containing 0.9 a of sodium chloride at pH 4.5 immediately before administration. Diabetes was confirmed by measurement

15 of blood glucose levels.

As can be seen in Table 1, and unlike control mice, whose blood glucose values increased throughout the course of the experiment, diabetic transgenic animals

20 showed normalization in blood glucose levels after 2 months after treatment with Stz, as well as a recovery of serum insulin levels due to the regeneration of pancreatic β cells.

25

Claims

R E I V I N D I C A C I O N E S 1. Chimeric gene that uses the insulin-like growth factor gene or cDNA of type I (IGF-I) directed by a promoter or fusion of promoters that allows the expression of IGF-I in the pancreas. 2. Chimeric gene according to claim 1, containing a promoter or fusion of romotores ^{p r} ue allow regulated expression of the growth factor similar to insulin type I (IGF-I ^ in exocrinc pancreas. 3. Chimeric gene according to reivindicacióp ^ containing a promoter or fusion of promoters which ^p rmiten ι_a regulated expression of the growth factor similar to insulin type I (IGF-I) in any of l: fi nnq -

4. Chimeric gene according to claim 1, nnri oi _o H " ^• • ^• n Qc rrπ o

allow regulated expression of the growth factor ^c imil ^a r ^to the insulin of ti ^ or I 'IGF-1 ¹ * ^α n róiπia R. pancreatic. . The ^q imeric r ^ la r ° ivindi "' ^:::, _' ^~ '^~; ^ ^η rnn pη-i __ -ι l Λ - - r ηr-nτnηi-nι ofn ς - • ó-n H <___ mπ ^ π Qc; mine allow regulated expression of insulin-like growth factor type I (IGF-I) in β cells ^ ncreá - * - icas being this promoter the ^ romotor of ^ a _ T_n_ ~ ςnι _-_ 1 ix pd. 6. An expression vector allows ex ^p SART the σuimérico differ according to any of claims 1 to 5 in pancreas. 7. Expression vector according to claim 6, this vector being a plasmid. 8. Expression vector according to claim 6, this vector being a viral vector. 9. Expression vector according to claim 6, this vector being a non-viral vector. 10. Viral vector according to claim 8, this vector being a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a Sindbis viral vector, a lentiviral vector, or a vector derived from herpes virus. 11. Pancreatic cell expressing the chimeric gene c rπ'i'ri npli π na ri or I pc; τ_- ϊ 7 -i 'n rH

1 _s R 12. Pancreatic cell according to claim 11, wherein the chimeric gene has been introduced by a vector according to any of claims 6 to 1C, for use in the development of therapeutic approaches for diabetes mellitus. 13. Use of a chimeric gene according to any of claims 1 to 5, for use in the development of therapeutic approaches for diabetes m ° lii ^ ^~ us 14. Use of an expression vector according to any of claims 6 to 10, for its πtτ_ι -i nation er_ the development of therapeutics for diabetes mellitus.