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Link:  Pharm/Biotech Resources


Title:  Production of pancreatic islet cells and delivery of insulin

United States Patent:  6,967,019

Issued:  November 22, 2005

Inventors:  German; Michael S. (Daly City, CA)

Assignee:  The Regents of the University of California (Oakland, CA)

Appl. No.:  817360

Filed:  March 20, 2001

Abstract

The present invention relates to the production of islet cells and insulin in a subject by providing for expression of an islet transcription factors in the pancreas of the subject, by for example, introduction of nucleic acid encoding the transcription factor neurogenin3 or a factor that induces neuorgenin3 expression. The present invention also relates to methods for using a islet transcription factor gene and the islet transcription factor polypeptide to alter cellular differentiation in culture or in vivo to produce new β-cells to treat patients with diabetes mellitus.

SUMMARY OF THE INVENTION

The present invention relates to the production of islet cells and insulin in a subject by providing for expression of an islet transcription factors in the pancreas of the subject, by for example, introduction of nucleic acid encoding the transcription factor neurogenin3 or a factor that induces neuorgenin3 expression. The present invention also relates to methods for using a islet transcription factor gene and the islet transcription factor polypeptide to alter cellular differentiation in culture or in vivo to produce new β-cells to treat patients with diabetes mellitus.

A primary object of the invention is to provide for the production of islet cells in the pancreas of a subject.

Another object of the invention is to provide for the production of insulin in a subject by inducing the formation of functional β-cells.

Another object of the invention is to provide a method for using the islet transcription factor genes to alter cellular differentiation in culture or in vivo to produce new β-cells to treat patients with diabetes mellitus.

Another object of the invention is to provide for production of islet cells for ex vivo therapy, e.g. production of islet transcription factor expressing cells for transplantation into a subject.

These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the invention more fully set forth below.

DETAILED DESCRIPTION OF THE INVENTION

Overview of the Invention

The present invention is based upon the discovery that the introduction of a polynucleotide sequence encoding an islet transcription factor into a pancreatic cell or other appropriate cell induces the production of cells having the phenotype of pancreatic islet cells, including insulin-producing β-cells. This discovery in turn is based on the discovery that providing for increased neurogenin3 (Ngn3) activity in a mature pancreatic cell (a non-beta cell), provides for development of the non-beta pancreatic cell into a cell with the pancreatic beta cell phenotype (e.g., production of insulin).

An increase in Ngn3 activity can be accomplished by, for example, introducing an Ngn3-encoding polynucleotide into a cell to provide for Ngn3 expression (which may be in addition to endogenous Ngn3 expression in the cell); providing for increased levels of expression of a positive regulator of Ngn3 (e.g., by introducing a polynucleotide encoding a transcription factor that positively regulates Ngn3 expression, or otherwise increasing activity or expression of such Ngn3 positive regulators); inhibiting activity (e.g., by inhibiting expression) of a negative regulator or inhibitor of Ngn3 expression or activity); increasing expression of a downstream effector which is positively regulated by Ngn3; and other variations that will be readily apparent to the ordinarily skilled artisan upon reading the present specification. Modulating of transcription factor activity (e.g., increasing Ngn3 activity or decreasing activity of an inhibitor of Ngn3 expression) can also be accomplished by use of signaling molecules (receptors, ligands, intracellular effectors), as well as synthetic and natural small molecule regulators of the pathway.

The invention generally involves providing for increased expression of at least one islet transcription factor selected from the neurogenic basic helix-loop-helix factors (bHLH) including the neurogenins (neurogenin1/NEUROG1/MATH4C/NeuroD3, neurogenin2/NEUROG2/MATH4A or neurogenin3/NEUROG3/MATH4B), the neuroD factors (NeuroD1/BETA2/BHF1, NeuroD2/NDRF, MATH2/NEX1/DLX3, NeuroD4/Math3), the Mash factors (Mash1 and Mash2), and the atonal-related factors (MATH1/ATOH1), as well as combinations thereof or combinations with other genes, to provide for induction of pancreatic beta cells.

FIG. 10 is a schematic representation of an alignment tree for neuroendorcrine bHLH proteins, plus myoD (a myogenic class B bHLH protein, and E47 (class A (ubiquitous) bHLH protein). All are based on mouse amino acid sequences. The sequences were aligned using the multiple sequence alignment algorithm Clustal-W as supplied in the MacVector6.5.1 sequence analysis program (Oxford Molecular). The definition of class A and B is based on the classification of Murre et al. (Murre, et al. (1989) Cell 58(3), 537-44).

Islet transcription factors are involved in the differentiation and development of islet cells. Islet transcription factors include members of the class B basic helix-loop-helix (bHLH) family of transcription factors, a family of factors known to regulate growth and differentiation of numerous cell types. Islet cells and the developing pancreas express a broad group of class B bHLH genes, among the most abundant being Ngn3, NeuroD1/BETA2, Mash1 and NeuroD4/Math3. NeuroD1 is also known as BETA2 and has been shown to be involved in the early differentiation of islet cells and the regulation of insulin transcription in pancreatic beta cells. Neurogenin3 activates NeuroD1/BETA2 during pancreatic development and therefore neurogenin3 lies upstream of neuroD1 in the hierarchy of islet transcription factors activated during islet cell differentiation. Ngn3 is expressed in islet cell progenitors and functions as a pro-endocrine gene, driving islet cell differentiation. Ngn3 is expressed early on in the development of all four islet cell types and is involved in the regulation of other islet transcription factors such as Pax4 and Nkx2.2 as well as NeuroD1/BETA1. Early and ectopic expression of Ngn3 can cause early and ectopic differentiation of islet cells. Other islet transcription factors also include non-bHLH factors such as the homeodomain factors, e.g. Nkx2.2 and Nkx6.1. These factors are immediately upstream or downstream of ngn3 and are involved in islet cell development. The pou-homeodomain factor HNF1 and the winged-helix factor HNF3 lie upstream of ngn3, and along with the cut-homeodomain factor HNF6 have been implicated in islet cell differentiation and are further examples of islet transcription factors in accordance to the present invention.

While Ngn3 is referred to throughout the specification, such reference is not intended to be limiting. Rather Ngn3 is only exemplary of islet transcription factors useful in the invention, and reference to it alone is for clarity and ease in review of the specification.

Induction of Beta-Cell Development

Pancreatic beta-cells can be produced from non-beta cell pancreatic cells by providing for production of an islet transcription factor in a pancreatic cell either in vivo (e.g., by administration of islet transcription factor-encoding nucleic acid (e.g., RNA or DNA) to the pancreas of a subject, e.g., by introduction of nucleic acid into a lumen of a pancreatic duct), or in vitro, e.g., by contacting a target cell (e.g., an isolated, non-beta, pancreatic cell) with islet transcription factor-encoding nucleic acid (e.g., RNA or DNA) in culture (which cells are then cultured, expanded, and transplanted into a subject).

In one embodiment of particular interest, beta cells are produced by providing for expression of neurogenin3 (Ngn3) at a level sufficient to induce the beta cell phenotype in the target cell. Expression of Ngn3 in the target cell can be accomplished in a variety of ways. For example, in one embodiment, Ngn3 expression is accomplished by introduction of Ngn3-encoding nucleic acid (e.g., DNA or RNA) to provide for expression of the encoded Ngn3 polypeptide in the target cell). In another embodiment Ngn3 expression is induced by introduction of a gene encoding a protein that provides for induction of Ngn3 expression (e.g., expression of an "upstream" positive regulator of Ngn3 expression in the target cell). In another embodiment, Ngn3 expression is accomplished by introduction of a gene encoding a protein that inhibits activity (e.g., function or expression) a negative regulator of Ngn3 expression. In another embodiment Ngn3 expression is induced by introduction of a small molecule that provides for induction of Ngn3 expression (e.g., a small molecule pharmaceutical that induces Ngn3 expression in the target cell). In addition, production of pancreatic beta cells of the invention can also be accomplished by providing for production of factors induced by Ngn3.

As will be readily appreciated by the ordinarily skilled artisan upon reading the present disclosure, Ngn3 expression can be accomplished by providing for any combination of these approaches. For example, the invention also encompasses providing for expression in the target cell of both an Ngn3-encoding nucleic acid as well as a positive regulator of an endogenous Ngn3 gene; providing for expression of an introduced Ngn3 nucleic acid as well as an inhibitor of a negative regulator of an endogenous Ngn3 and introduced Ngn3 sequence; and the like. In general, any combination of the approaches that provide for Ngn3 activity by, for example, providing for expression of Ngn3 per se (by introduction of Ngn3-encoding nucleic acid or providing for expression of endogenous Ngn3) and/or by providing of production of factors "downstream" of Ngn3 that are normally produced as a result of Ngn3 expression, are within the scope of the present invention. Positive regulators of Ngn3 expression include, but are not necessarily limited to HNF1, HNF3, and HNF6.

In general, factors that provide for production of Ngn3 activity in a target cell are referred to herein as "islet transcript factors." As noted above "islet transcription factor" is meant any transcription factor involved in the differentiation and/or development of islet cells, the expression of which contributes to the production of a cell having an islet cell phenotype, e.g., a cell that produces insulin or other markers characteristic of islet cells, as well as functionally equivalent homologues. Of particular interest are the class B basic helix-loop-helix (bHLH) transcription factors involved in the development of islet cells, which include the neurogenins (neurogenin1, neurogenin2 and neurogenin3), the neuroD factors (NeuroD1/BETA2, neuroD2, and NeuroD4/Math3) and the Mash factor, Mash1, as well as functionally equivalent homologues of these transcription factors. Reference to Ngn3 herein is for clarity, and is not meant to be limiting, but rather to provide a reference point in a regulatory pathway that leads to pancreatic beta cell development.

In addition, induction of the activity of the Ngn3 pathway can be accomplished using naturally occurring or synthetic molecules other than nucleic acid. For example, Ngn3 activity can be induced by using a synthetic molecule that promotes Ngn3 expression, e.g., by inhibiting activity of a negative regulator of Ngn3 expression. Inhibitory transcription factors of Ngn3 expression include, but are not necessarily limited to HES1. Negative signally pathways that inhibit Ngn3 expression include, but are not necessarily limited to, the Notch pathway.

Islet Transcription Factor Nucleic Acids

The term "islet transcription factor gene" is used to designate both transcription factors that are expressed in pancreatic islet cells, and also transcription factors that are involved in the development, differentiation, or formation of islet cells. The term "islet transcription factor gene" is also intended to mean the open reading frame encoding specific islet transcription factor polypeptides, introns, and adjacent 5′ and 3′ non-coding nucleotide sequences involved in the regulation of expression, up to about 10 kb beyond the coding region, but possibly further in either direction. The DNA sequences encoding an islet transcription factor may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.

The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons (e.g., sequences encoding open reading frames of the encoded polypeptide) and 3′ and 5′ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the polypeptide of interest.

A islet transcription factor genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It may further include the 3′ and 5′ untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 10 kb, but possibly more, of flanking genomic DNA at either the 5′ or 3′ end of the transcribed region. The genomic DNA may be isolated as a large fragment of 100 kbp or more, or as a smaller fragment substantially free of flanking chromosomal sequence.

The sequence of this 5′ region, and further 5′ upstream sequences and 3′ downstream sequences, may be utilized for promoter elements, including enhancer binding sites, that provide for expression in tissues where the islet transcription factor is expressed. The sequences of the islet transcription factor promoter elements of the invention can be based on the nucleotide sequences of any species (e.g., mammalian or non-mammalian (e.g., reptiles, amphibians, avian (e.g., chicken)), particularly mammalian, including human, rodenti (e.g., murine or rat), bovine, ovine, porcine, murine, or equine, preferably mouse or human) and can be isolated or produced from any source whether natural, synthetic, semi-synthetic or recombinant.

The nucleic acid compositions used in the subject invention may encode all or a part, usually at least substantially all, of the islet transcription factor polypeptides as appropriate. Fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least about ten contiguous nucleotides, usually at least about 15 nt, more usually at least about 18 nt to about 20 nt, more usually at least about 25 nt to about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used. The exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art. It is preferable to choose a pair of primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other.

The islet transcription factor genes are isolated and obtained in substantial purity, generally as other than an intact mammalian chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a sequence encoding an islet transcription factor or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

The sequence of the islet transcription factor, including flanking promoter regions and coding regions, may be mutated in various ways known in the art to generate targeted changes in promoter strength, sequence of the encoded protein, etc. The DNA sequence or product of such a mutation will be substantially similar to the sequences provided herein, i.e. will differ by at least one nucleotide or amino acid, respectively, and may differ by at least two, or by at least about ten or more nucleotides or amino acids. In general, the sequence changes may be substitutions, insertions or deletions. Deletions may further include larger changes, such as deletions of a domain or exon. It should be noted that islet transcription factor sequences are conversed mainly within the bHLH domain, and regions outside this domain may not be as well-conserved, and may even be remarkably poorly conserved, between, for example, rat, mouse, and humans. Thus islet transcription factors can tolerate more nucleotide and amino acid residue changes outside of the bHLH domain and retain function to a much greater extent than changes made within the bHLH domain. Such modified islet transcription factor sequences can be used, for example, to generate vectors for introduction into target cells for the purpose of producing islet cells.

Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al., 1993 Biotechniques 14:22; Barany, 1985 Gene 37:111-23; Colicelli et al., 1985 Mol Gen Genet 199:537-9; and Prentki et al., 1984 Gene 29:303-13. Methods for site specific mutagenesis can be found in Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, CSH Press, pp. 15.3-15.108; Weiner et al., 1993 Gene 126:35-41; Sayers et al., 1992 Biotechniques 13:592-6; Jones and Winistorfer, 1992 Biotechniques 12:528-30; Barton et al., 1990 Nucleic Acids Res 18:7349-55; Marotti and Tomich, 1989 Gene Anal Tech 6:67-70; and Zhu 1989 Anal Biochem 177:120-4.

An islet transcription factor of particular interest in the present invention is a member of the neurogenin transcription factor family, e.g., neurogenin 1 (Ngn1), neurogenin2 (Ngn2), neurogenin 3 (Ngn3), with Ngn3 being of particular interest. The nucleotide and amino acid sequences of human Ngn3 are provided in the Sequence Listing as SEQ ID NOS: 1 and 2, respectively. The nucleotide and amino acid sequences of human Ngn1 are available at GenBank accession number XM003834 and NM006161. The nucleotide and amino acid sequence of human Ngn2 are available at GenBank accession number AF303002.

It should be noted that transcription factors which act either "upstream" of ngn3 (and therefore activate ngn3 expression) or "downstream" of Ngn3, that lead to development of the islet cell phenotype, are also contemplated for use in the present invention.

Neurogenin3 by itself, is sufficient to force undifferentiated pancreatic epithelial cells to become islet cells. Since neurogenin3 expression determines which precursor cells will differentiate into islet cells, the signals that regulate neurogenin3 expression are also involved in islet cell formation. Although 2.7 kb of the ngn3 promoter is sufficient to direct expression correctly in transgenic mice, distal sequences have been shown to greatly enhance the expression of ngn3. This distal promoter region contains a cluster of binding sites for pancreatic transcription factors such as, HNF6, HNF1α, and HNF3β. These pancreatic transcription factors have been found to regulate ngn3 gene expression and thereby are also involved in the control of islet cell formation. These signals may be useful in generating new islet cells for patients with diabetes mellitus.

In another embodiment, the islet transcription factor is human NeuroD1/BETA2 gene, which is available, with the corresponding human NeuroD1/BETA2 amino acid sequence, at GenBank accession number NM002500.

The human Mash1gene and the corresponding amino acid sequence are available at GenBank accession number XM006688.

The human NeuroD4/Math3 gene and the corresponding human NeuroD4/Math3 amino acid sequence are available at GenBank accession number AF203901.

Constructs for Delivery of Islet Transcription Factor Nucleic Acid

Where the islet transcription factor nucleic acid to be delivered is DNA, any construct having a promoter (e.g., a promoter that is functional in a eukaryotic cell) operably linked to a DNA of interest can be used in the invention. The constructs containing the DNA sequence (or the corresponding RNA sequence) which may be used in accordance with the invention may be any eukaryotic expression construct containing the DNA or the RNA sequence of interest. For example, a plasmid or viral construct (e.g. adenovirus) can be cleaved to provide linear DNA having ligatable termini. These termini are bound to exogenous DNA having complementary-like ligatable termini to provide a biologically functional recombinant DNA molecule having an intact replicon and a desired phenotypic property. Preferably the construct is capable of replication in eukaryotic and/or prokaryotic hosts (viruses in eukaryotic, plasmids in prokaryotic), which constructs are known in the art and are commercially available.

The constructs can be prepared using techniques well known in the art. Likewise, techniques for obtaining expression of exogenous DNA or RNA sequences in a genetically altered host cell are known in the art (see, for example, Kormal et al., Proc. Natl. Acad. Sci. USA, 84:2150-2154, 1987; Sambrook et al. Molecular Cloning: a Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., each of which are hereby incorporated by reference with respect to methods and compositions for eukaryotic expression of a DNA of interest).

In one embodiment, the DNA construct contains a promoter to facilitate expression of the DNA of interest within a pancreatic cell. The promoter may be a strong, viral promoter that functions in eukaryotic cells such as a promoter from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), or adenovirus. More specifically, exemplary promoters include the promoter from the immediate early gene of human CMV (Boshart et al., Cell 41:521-530, 1985) and the promoter from the long terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781, 1982). Of these two promoters, the CMV promoter is presently preferred as it provides for higher levels of expression than the RSV promoter.

Alternatively, the promoter used may be a strong general eukaryotic promoter such as the actin gene promoter. In one embodiment, the promoter used may be a tissue-specific promoter. For example, the promoter used in the construct may be a pancreas specific promoter, a duct cell specific promoter or a stem cell specific promoter. The constructs of the invention may also include sequences in addition to promoters which enhance expression in the target cells.

In another embodiment, the promoter is a regulated promoter, such as a tetracycline-regulated promoter, expression from which can be regulated by exposure to an exogenous substance (e.g., tetracycline.).

Other components such as a marker (e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) or β-galactosidase) aid in selection or identification of cells containing and/or expressing the construct, an origin of replication for stable replication of the construct in a bacterial cell (preferably, a high copy number origin of replication), a nuclear localization signal, or other elements which facilitate production of the DNA construct, the protein encoded thereby, or both.

For eukaryotic expression, the construct should contain at a minimum a eukaryotic promoter operably linked to a DNA of interest, which is in turn operably linked to a polyadenylation signal sequence. The polyadenylation signal sequence may be selected from any of a variety of polyadenylation signal sequences known in the art. An exemplary polyadenylation signal sequence is the SV40 early polyadenylation signal sequence. The construct may also include one or more introns, where appropriate, which can increase levels of expression of the DNA of interest, particularly where the DNA of interest is a cDNA (e.g., contains no introns of the naturally-occurring sequence). Any of a variety of introns known in the art may be used (e.g., the human β-globin intron, which is inserted in the construct at a position 5′ to the DNA of interest).

In an alternative embodiment, the nucleic acid delivered to the cell is an RNA encoding an islet transcription factor. In this embodiment, the RNA is adapted for expression (i.e., translation of the RNA) in a target cell. Methods for production of RNA (e.g., mRNA) encoding a protein of interest are well known in the art, and can be readily applied to the product of RNA encoding islet transcription factors useful in the present invention.

Delivery of Islet Transcription Factor-Encoding Nucleic Acid

Delivery of islet transcription factor-encoding nucleic acid can be accomplished using a viral or a non-viral vector. In one embodiment the nucleic acid is delivered within a viral particle, such as an adenovirus. In another embodiment, the nucleic acid is delivered in a formulation comprising naked DNA admixed with an adjuvant such as viral particles (e.g., adenovirus) or cationic lipids or liposomes. An "adjuvant" is a substance that does not by itself produce the desired effect, but acts to enhance or otherwise improve the action of the active compound. The precise vector and vector formulation used will depend upon several factors, such as the size of the DNA to be transferred, the delivery protocol to be used, and the like. Exemplary non-viral and viral vectors are described in more detail below.

I. Viral Vectors

In general, viral vectors used in accordance with the invention are composed of a viral particle derived from a naturally-occurring virus which has been genetically altered to render the virus replication-defective and to deliver a recombinant gene of interest for expression in a target cell in accordance with the invention.

Numerous viral vectors are well known in the art, including, for example, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus vectors. Adenovirus and AAV are usually preferred viral vectors since these viruses efficiently infect slowly replicating and/or terminally differentiated cells. The viral vector may be selected according to its preferential infection of the cells targeted.

Where a replication-deficient virus is used as the viral vector, the production of infectious virus particles containing either DNA or RNA corresponding to the DNA of interest can be achieved by introducing the viral construct into a recombinant cell line which provides the missing components essential for viral replication. In one embodiment, transformation of the recombinant cell line with the recombinant viral vector will not result in production or substantial production of replication-competent viruses, e.g., by homologous recombination of the viral sequences of the recombinant cell line into the introduced viral vector. Methods for production of replication-deficient viral particles containing a nucleic acid of interest are well known in the art and are described in, for example, Rosenfeld et al., Science 252:431-434, 1991 and Rosenfeld et al., Cell 68:143-155, 1992 (adenovirus); U.S. Pat. No. 5,139,941 (adeno-associated virus); U.S. Pat. No. 4,861,719 (retrovirus); and U.S. Pat. No. 5,356,806 (vaccinia virus). Methods and materials for manipulation of the mumps virus genome, characterization of mumps virus genes responsible for viral fusion and viral replication, and the structure and sequence of the mumps viral genome are described in Tanabayashi et al., J. Virol. 67:2928-2931, 1993; Takeuchi et al., Archiv. Virol., 128:177-183, 1993; Tanabayashi et al., Virol. 187:801-804, 1992; Kawano et al., Virol., 179:857-861, 1990; Elango et al., J. Gen. Virol. 69:2893-28900, 1988.

II. Non-viral Vectors

The nucleic acid of interest may be introduced into a cell using a non-viral vector. "Non-viral vector" as used herein is meant to include naked DNA (e.g., DNA not contained within a viral particle, and free of a carrier molecules such as lipids), chemical formulations comprising naked nucleic acid (e.g., a formulation of DNA (and/or RNA) and cationic compounds (e.g., dextran sulfate, cationic lipids)), and naked nucleic acid mixed with an adjuvant such as a viral particle (e.g., the DNA of interest is not contained within the viral particle, but the formulation is composed of both naked DNA and viral particles (e.g., adenovirus particles) (see, e.g., Curiel et al. 1992 Am. J. Respir. Cell Mol. Biol. 6:247-52). Thus "non-viral vector" can include vectors composed of nucleic acid plus viral particles where the viral particles do not contain the DNA of interest within the viral genome.

In one embodiment, the formulation comprises viral particles which are mixed with the naked DNA construct prior to administration. About 108 to about 1010 viral particles (preferably about 1×1010 to about 5×1010, more preferably about 3×1010 particles) are mixed with the naked DNA construct (about 5 μg to 50 μg DNA, more preferably about 8 μg to 25 μg DNA) in a total volume of about 100 μl. Preferably the viral particles are adenovirus particles (Curiel et al., 1992 supra).

Alternatively or in addition, the nucleic acid can be complexed with polycationic substances such as poly-L-lysine or DEAC-dextran, targeting ligands, and/or DNA binding proteins (e.g., histones). DNA- or RNA-liposome complex formulations comprise a mixture of lipids which bind to genetic material (DNA or RNA) and facilitate delivery of the nucleic acid into the cell. Liposomes which can be used in accordance with the invention include DOPE (dioleyl phosphatidyl ethanol amine), CUDMEDA (N-(5-cholestrum-3-.beta.-ol 3-urethanyl)-N′,N′-dimethylethylene diamine).

For example, the naked DNA can be administered in a solution containing Lipofectin™ (LTI/BRL) at a concentrations ranging from about 2.5% to 15% volume: volume, preferably about 6% to 12% volume:volume. Preferred methods and compositions for formulation of DNA for delivery according to the method of the invention are described in U.S. Pat. No. 5,527,928, the disclosure of which is incorporated herein by reference.

The nucleic acid of interest can also be administered as a chemical formulation of DNA or RNA coupled to a carrier molecule (e.g., an antibody or a receptor ligand) which facilitates delivery to host cells for the purpose of altering the biological properties of the host cells. By the term "chemical formulations" is meant modifications of nucleic acids which allow coupling of the nucleic acid compounds to a carrier molecule such as a protein or lipid, or derivative thereof. Exemplary protein carrier molecules include antibodies specific to the cells of a targeted pancreatic cell or receptor ligands, e.g., molecules capable of interacting with receptors associated with a cell of a targeted pancreatic cell.

Production of Islet Transcription Factor Polypeptides and Antibodies that Specifically Bind such Polypeptides

Nucleic acid encoding Ngn3 or other islet transcription factors of interest may be employed to synthesize full-length polypeptides or fragments thereof, particularly fragments corresponding to functional domains; DNA binding sites; etc.; and including fusions of the subject polypeptides to other proteins or parts thereof. Accordingly, the polynucleotides and polypeptides suitable for use in the invention include, without limitation, islet transcription factor polypeptides and polynucleotides found in primates, rodents, canines, felines, equines, nematodes, yeast and the like, and the natural and non-natural variants thereof.

The islet transcription factor polypeptides can be used for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, and larger fragments or the entire protein allow for the production of antibodies over the surface of the polypeptide. Antibodies may be raised to the wildtype or variant forms of the polypeptide. Antibodies may be raised to isolated peptides corresponding to these domains, or to the native protein, e.g. by immunization with cells expressing the polypeptide of interest, immunization with liposomes having a polypeptide of interest inserted in the membrane, etc.

Antibodies are prepared in accordance with conventional ways, where the expressed polypeptide or protein is used as an immunogen, by itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like. For further description, see Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1988.

Antibodies that specifically bind islet transcription factors can be utilized to detect cells expressing a recombinant islet transcription factor such as Ngn3 (e.g., prior to transplantation).

Production of Islet Cells by Expression of Transcription Factor-Encoding Nucleic Acids

Islet cells can be produced according to the invention in a variety of ways. In general, the invention involves stimulating the production of an islet transcription factor. In an embodiment of particular interest, the invention involves enhancing islet transcription factor activity by introducing a nucleic acid encoding an islet transcription factor into a cell, usually a pancreatic cell.

I. Production of Islet Cells In Vitro by Introduction of an Islet Transcription Factor-Encoding Nucleic Acid

Nucleic acid encoding an islet transcription factor (e.g., Ngn3) can be introduced into a cell in vitro to accomplish expression in the cell to provide for at least transient expression. The cells into which the nucleic acid is introduced can be differentiated epithelial cells (e.g., pancreatic cells, gut cells, hepatic cells or duct cells), pluripotent adult or embryonic stem cells, or any mammalian cell capable of developing into β cells or cells capable of expression of insulin in vitro following expression of an islet transcription factor-encoding nucleic acid. The cell is subsequently implanted into a subject having a disorder characterized by a deficiency in insulin, which disorder is amenable to treatment by islet cell replacement therapy. In one embodiment, the host cell in which Ngn3 expression is provided and which is implanted in the subject is derived from the individual who will receive the transplant (e.g., to provide an autologous transplant). For example, in a subject having Type 1 diabetes, pluripotent stem cells, hepatic cells, gut cells or pancreatic cells can be isolated from the affected subject, the cells modified to express Ngn3-encoding DNA, and the cells implanted in the affected subject to provide for insulin production, or the transformed cells cultured so as to facilitate development of the cells into insulin-producing β-cells. Alternatively, pluripotent stem cells, hepatic cells, gut cells or pancreatic cells from another subject (the "donor") could be modified to express Ngn3-encoding DNA, and the cells subsequently implanted in the affected subject to provide for insulin production, or the transformed cells cultured so as to facilitate development of the cells into insulin-producing β-cells.

Introduction of nucleic acid into the cell in vitro can be accomplished according to methods well known in the art (e.g., through use of electroporation, microinjection, lipofection infection with a recombinant (preferably replication-deficient) virus, and other means well known in the art). The nucleic acid is generally operably linked to a promoter that facilitates a desired level of polypeptide expression (e.g., a promoter derived from CMV, SV40, adenovirus, or a tissue-specific or cell type-specific promoter). Transformed cells containing the recombinant nucleic acid can be selected and/or enriched via, for example, expression of a selectable marker gene present in the introduced construct or that is present on a nucleic acid that is co-transfected with the construct. Typically selectable markers provide for resistance to antibiotics such as tetracycline, hygromycin, neomycin, and the like. Other markers can include thymidine kinase and the like. Other markers can include markers that can be used to identify expressing cells, such as beta-galactosidase or green florescent protein.

Expression of the introduced nucleic acid in the transformed cell can be assessed by various methods known in the art. For example, expression of the introduced gene can be examined by Northern blot to detect mRNA which hybridizes with a DNA probe derived from the relevant gene. Those cells that express the desired gene can be further isolated and expanded in in vitro culture using methods well known in the art. The host cells selected for transformation will vary with the purpose of the ex vivo therapy (e.g., insulin production), the site of implantation of the cells, and other factors that will vary with a variety of factors that will be appreciated by the ordinarily skilled artisan.

The transformed cell can also be examined for the development of an islet cell phenotype. For example, expression of insulin could be detected by PCR, northern blot, immunocytochemistry, western blot. RIA or ELISA Alternatively a marker gene such as green florescent protein or an antibiotic resistance gene operatively linked to an islet specific promoter such as the insulin gene promoter could be used for identification or selection of differentiated islet cells. Methods for engineering a host cell for expression of a desired gene product(s) and implantation or transplantation of the engineered cells (e.g., ex vivo therapy) are known in the art (see, e.g., Gilbert et al. 1993 "Cell transplantation of genetically altered cells on biodegradable polymer scaffolds in syngeneic rats," Transplantation 56:423-427). For expression of a desired gene in exogenous or autologous cells and implantation of the cells (e.g., islet cells) into pancreas, see, e.g., Docherty 1997 "Gene therapy for diabetes mellitus," Clin Sci (Colch) 92:321-330; Hegre et al. 1976 "Transplantation of islet tissue in the rat," Acta Endocrinol Suppl (Copenh) 205:257-281; Sandler et al. 1997 "Assessment of insulin secretion in vitro from microencapsulated fetal porcine islet-like cell clusters and rat, mouse, and human pancreatic islets," Transplantation 63:1712-1718; Calafiore 1997 "Perspectives in pancreatic and islet cell transplantation for the therapy of IDDM," Diabetes Care 20:889-896; Kenyon et al. 1996 "Islet cell transplantation: beyond the paradigms," Diabetes Metab Rev 12:361-372; Sandler; Chick et al. 1977 Science "Artificial pancreas using living beta cells: effects on glucose homeostasis in diabetic rats," 197:780-782. In general, the cells can be implanted into the pancreas, or to any practical or convenient site, e.g., subcutaneous site, liver, peritoneum.

Methods for transplanting islets cells are well known in the art, see, e.g., Hegre et al. 1976 "Transplantation of islet tissue in the rat," Acta Endocrinol Suppl (Copenh) 205:257-281; Sandler et al. 1997 "Assessment of insulin secretion in vitro from microencapsulated fetal porcine islet-like cell clusters and rat, mouse, and human pancreatic islets," Transplantation 63:1712-1718; Calafiore 1997 "Perspectives in pancreatic and islet cell transplantation for the therapy of IDDM," Diabetes Care 20:889-896; Kenyon et al. 1996 "Islet cell transplantation: beyond the paradigms," Diabetes Metab Rev 12:361-372; Sandler; Chick et al. 1977 Science "Artificial pancreas using living beta cells: effects on glucose homeostasis in diabetic rats," 197:780-782.

In general, after expansion of the transformed cells in vitro, the cells are implanted into the mammalian subject by methods well known in the art. The number of cells implanted is a number of cells sufficient to provide for expression of levels of insulin sufficient to lower blood glucose levels. The number of cells to be transplanted can be determined based upon such factors as the levels of polypeptide expression achieved in vitro, and/or the number of cells that survive implantation. The transformed cells are implanted in an area of dense vascularization such as the liver, and in a manner that minimizes surgical intervention in the subject. The engraftment of the implant of transformed cells is monitored by examining the mammalian subject for classic signs of graft rejection, i.e., inflammation and/or exfoliation at the site of implantation, and fever, and by monitoring blood glucose levels.

The transplantation method described above is not limited to the expression of nerougenin3. Engineering a host cell for expression of other islet transcription factors in the differentiation cascade, such as islet factors and in particular, NeuroD1/BETA2 may be beneficial to subjects with insulin deficiencies.

II. In Vivo Development of Islet Cells and Production of Insulin in the Pancreas

Islet transcription factor-encoding nucleic acid can be delivered directly to a subject to provide for islet transcription factor expression in a target cell (e.g., a pancreatic cell, gut cell, liver cell, or other organ cell capable of expressing an islet transcription factor and providing production of insulin), thereby promoting development of the cell into an insulin-producing cell (e.g., in pancreas) or to cure a defect in islet transcription factor expression in the subject. Methods for in vivo delivery of a nucleic acid of interest for expression in a target cell are known in the art. For example, in vivo methods of gene delivery normally employ either a biological means of introducing the DNA into the target cells (e.g., a virus containing the DNA of interest) or a mechanical means to introduce the DNA into the target cells (e.g., direct injection of DNA into the cells, liposome fusion, or pneumatic injection using a gene gun).

In general, the transformed cells expressing the protein encoded by the DNA of interest produce a therapeutically effective amount of the protein to produce islet cells, in particular β-cells in the mammalian patient. In one embodiment, the DNA of interest encodes an islet transcription factor such as Neurogenin1, Neurogenin2, Neurogenin3, NeuroD1/BETA2, Mash1 or NeuroD4/Math3 (with Ngn3 being of particular interest), and the DNA of interest is operably linked to a promoter, which may be heterologous or endogenous to the transcription factor.

In general terms, the delivery method comprises introducing the nucleic of interest-containing vector into a pancreatic cell. By way of example, DNA of interest-containing vector may comprise either a viral or non-viral vector (including naked DNA), which is introduced into the pancreas in vivo via the duct system. Intraductal administration can be accomplished by cannulation by, for example, insertion of the cannula through a lumen of the gastrointestinal tract, by insertion of the cannula through an external orifice, or insertion of the cannula through the common bile duct. Retrograde ductal administration may be accomplished in the pancreas by endoscopic retrograde chalangio-pancreatography (ECRP). Exemplary methods for accomplishing intraductal delivery to the pancreas are described in U.S. Pat. No. 6,004,944.

The precise amount of islet transcription factor-encoding nucleic acid administered will vary greatly according to a number of factors including the susceptibility of the target cells to transformation, the size and weight of the subject, the levels of protein expression desired, and the condition to be treated. The amount of nucleic acid and/or the number of infectious viral particles effective to infect the targeted tissue, transform a sufficient number of cells, and provide for production of a desired level of insulin can be readily determined based upon such factors as the efficiency of the transformation in vitro and the susceptibility of the targeted cells to transformation. For example, the amount of DNA introduced into the pancreatic duct of a human is, for example, generally from about 1 μg to about 750 mg, preferably from about 500 μg to about 500 mg, more preferably from about 10 mg to about 200 mg, most preferably about 100 mg. Generally, the amounts of DNA can be extrapolated from the amounts of DNA effective for delivery and expression of the desired gene in an animal model. For example, the amount of DNA for delivery in a human is roughly 100 times the amount of DNA effective in a rat.

Pancreatic cells modified according to the invention can facilitate sufficiently high levels of expression of a nucleic acid of interest, particularly where the nucleic acid delivered is DNA and the DNA of interest is operably linked to a strong eukaryotic promoter (e.g., CMV, MMTV). The expressed protein can induce islet cell and insulin production. Thus the methods of the invention are useful in treating a mammalian subject having a variety of insulin related conditions.

In the preferred embodiment, the encoded proteins are islet transcription factors from the class of basic helix-loop-helix (bHLH) proteins. For example, the expression of neurogeinin3 and/or NeuroD1/BETA2 may substantially induce the production of islet cells and insulin in mammals.

The actual number of transformed pancreatic cells required to achieve therapeutic levels of the protein of interest will vary according to several factors including the protein to be expressed, the level of expression of the protein by the transformed cells, the rate in which the protein induces islet cell production (in particular Beta cells), and the condition to be treated.

Regardless of whether the islet transcription factor-encoding nucleic acid is introduced in vivo or ex vivo, the nucleic acid (or islet cells produced in vitro or recombinant cells expressing the islet transcription factor nucleic acid that are to be transplanted for development into islet cells in vivo post-transplantation) can be administered in combination with other genes and other agents.

Assessment of Therapy

The effects of ex vivo or in vivo therapy according to the methods of the invention can be monitored in a variety of ways. Generally, a sample of blood from the subject can be assayed for, for example, levels of glucose, proinsulin, c-peptide, and insulin. Appropriate assays for detecting proinsulin, c-peptide, insulin and glucose in blood samples are well known in the art. Evidence for recurrent autoimmunity can be gauged by assaying for autoreactive T cells or for antibodies against islet proteins such as glutamic acid decarboxylase (GAD), or other autoantigens well known in the art.
 

Claim 1 of 21 Claims

1. A method for producing an insulin-producing cell in vitro, the method comprising:

introducing a nucleic acid molecule operably linked to a promoter into a cell in vitro, the nucleic acid molecule encoding a neuroendocrine class B basic helix-loop-helix (bHLH) transcription factor, said introducing being in an amount sufficient for production of the neuroendocrine bHLH transcription factor and production of an insulin-producing cell;

wherein said cell is a cultured gastrointestinal organ cell.

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