Therapeutic Nucleic Acids

Chapter 2Therapeutic Nucleic Acids II

2.1.8 Control of Therapeutic Gene Expression As already mentioned above, several of the current gene therapy applications based on the intracellular expression of protein-coding genes take advantage of strong constitutive promoters, such as the CMV IE gene promoter. 2.1.8 Control of Therapeutic Gene Expression In several situations, however, expression of the therapeutic protein (or regulatory RNA) must be limited to one specific cell type, or at least controlled in terms of levels, or restricted to a defined temporal period. For example, the genes coding for the different hemoglobin chains (alpha-and beta-globin in the adult) must be transcribed in a balanced manner2.1.8 Control of Therapeutic Gene Expression To avoid monomer aggregation and precipitation in the erythroblasts, causing apoptosis of these cellsin gene therapy of diabetes, insulin must be precisely produced in response to blood glucose concentration. Expression of some pro-apoptotic genes can be beneficial only if limited to cancer cells.2.1.8 Control of Therapeutic Gene Expression over-expression of some growth factors, including those inducing new blood vessel blood formation, might be detrimental expressed for excessively long periods.

2.1.8 Control of Therapeutic Gene Expression In several of these conditions, an optimal solution would be to use the natural promoter of the therapeutic gene to direct its expression in vivo. In most cases, however, this is not possible, since natural promoters are usually very large and thus cannot be accommodated in most viral vectors.

2.1.8.1 Tissue-Specific Promoters The first strategy to tackle the issue of regulated trans gene expression is to limit its transcription to the tissue of interest, using a promoter specific for this tissue and sufficiently short to be cloned in a gene transfer vector. 2.1.8.1 Tissue-Specific Promoters Examples of such transcriptional elements are the musde creatine-kinase (MCK) enhancer the beta-actin gene promoter for the skeletal muscle, the a-myosin heavy chain gene promoter (alpha MHC) for the heart, the insulin gene promoter for the pancreas, the albumin gene promoter for the liver, the transthyretin gene promoter for the retina, and so on. 2.1.8.1 Tissue-Specific Promoters An elegant manner to obtain the opposite effect, namely block transgene expression in a given tissue, is to act post-transcriptionally and exploit the presence, in several cell types, of natural microRNAs targeting various cellular genes .2.1.8.1 Tissue-Specific Promoters If the trans gene mRNA sequence is the target of a miRNA which can be obtained by cloning the miRNA recognition sequence downstream of the transgene coding region and before the polyadenylation site- protein expression will be selectively inhibited in the cell types expressing this miRNA while remaining unaffected in other cells. 2.1.8.1 Tissue-Specific Promoters This property can be used, for example, to block expression of a protein of interest in antigen-presenting cells (APCs), thus blocking its presentation to the cells of the immune system. 2.1.8.2 Inducible Promoters Besides the use of tissue-specific promoters, an alternative manner to regulate therapeutic gene expression is by using inducible promoters, in which transcription can be selectively activated. Some of these promoters are naturally present in the genome and direct gene expression under specific physiologic conditions. 2.1.8.2 Inducible Promoters Among these promoters are those of genes expressed in response to heavy metals- for example the methallothioein gene, heat shock proteins 70 and 90, hormonesMMT Virus long terminal repeat (LTR) promoter, sensitive to dexamethazone induction, and hypoxia -for example the VEGF angiogenic protein. 2.1.8.2 Inducible Promoters Most of these are natural promoters, however, cannot be easily used since transcriptional control is not very stringent, the level of induced expression are too low, they are activated in conditions that cannot be easily reproduced in a therapeutic setting, the pleiotropic undesired effects elicited by the stimulating agent itself (for example, high temperature or hormone administration).2.1.8.2 Inducible Promoters A very interesting category of promoters, in contrast, consists of a few synthetic promoters, the activity or which can be controlled phamacologically, by the administration of simple drugs. 2.1.8.2 Inducible Promoters These promoters respond to specific transcriptional activators that can be assigned to one of at least three classes: transcriptional activators regulated by small molecules; intracellular steroid hormone receptors; orsynthetic transcription factors in which dimerization is controlled by antibiotic rapamycin.

Inducible Promoters transcriptional activators controlled by small molecules This class of inducible regulators is based on the use of transcription factors that change their conformation (and are thus either activated or deactivated) upon binding one small chemical molecule. The prototype of this class of regulators is the Tet repressor (TetR), which, in E.coli regulates expression of the Tn10 operon genes. This operon encodes a system of transporters that determine exit of tetracycline from the bacterial cell, thus conferring resistance to this antibiotic.

2.1.8.2 Inducible Promoters transcriptional activators controlled by small molecules ln the absence of tetracycline, TetR binds a DNA sequence (the Tet operator, tetO) positioned upstream of the Tet operon and suppresses transcription. When tetracycline is instead present, this binds TetR and causes a confonnational change that blocks its interaction with tetO: in this manner, transcription of the Tn10 operon is de-repressed and the bacterium becomes resistant to the antibiotic. Inducible Promoters transcriptional activators controlled by small molecules This transcriptional control strategy was adapted to mammalian cells by generating a two-component system: on one hand, the gene of interest is placed under the transcriptional control of a minimal promoter - which dictates the localization of the transcription start site- with a series of TetO repetitions positioned upstream; on the other hand, another construct codes for a fusion protein between TetR and an RNA polymerase II transcriptional activator, such as the carboxyterminal domain of the herpes simplex virus type I (HSV-1) protein VPJ6 (the fusion protein between TetR and VPI6 is known as tTA).In the absence of tetracycline, tTA binds in multiple copies to the promoter and activates transcription (Fig. 2.3A). Inducible Promoters transcriptional activators controlled by small molecules When tetracycline or, better, its derivative doxycycline is present, transcription is instead switched off since the antibiotic binds the TetR moiety of tTA and the allosteric change determined by this interaction causes detachment of the hybrid transcription factor from the promoter. This system of transcriptional regulation is known as Tet-off; since the antibiotic blocks transcription.Inducible Promoters transcriptional activators controlled by small molecules A variant of this system was subsequently developed, based on the use of a four-amino-acid mutant of the TetR protein, known as reverse TetR (rTetR), which is unable to bind the promoter unless the antibiotic is present. When this protein is fused to VP16 to generate the reverse tTA (rtTA) regulator, transcription is switched on by the addition of the antibiotic (Tet-On system; Fig. 2.3B). Inducible Promoters transcriptional activators controlled by small moleculesA couple of these are derived from E. coli and exploit the antibiotic- dependent binding of other activators/repressors to their cognate DNA sites; these include the PIP transactivator binding the PIR binding site, which is regulated by pristinamycin, andthe E transactivator binding the ETR binding site, regulated by erythromycin. Alternatively, a system regulated by gaseous acetaldehyde was also developed, based on the AlcR transactivator derived from the fungus Aspergillus nidulans.

Intracellular receptors for steroid hormonesIntracellular receptors are proteins that, once complexed with their ligands, directly activate target gene expression in the nucleus. The prototypes of this class of molecules are the steroid hormone receptors. In particular, the estrogen and progesterone receptors are sequestered in the ceII cytosol by the Hsp90 protein, which masks their nuclear localization signal; binding to the respective hormones induces a conformational change that releases the receptors, allows their nuclear import, and thus activates target gene transcription.

Intracellular receptors for steroid hormonesTo exploit this system for the inducible expression of a desired gene, the natural receptor protein was engineered by fusing it with the DNA binding domain of the yeast transcription factor Gal4, in order to target this fusion protein towards synthetic promoters containing Gal4 binding sites, and by mutating its amino acid sequence to lower its affinity for the natural hormones and increase that for synthetic analogues, including estrogen and progesterone receptor. Intracellular receptors for steroid hormonesWhen cells are transfected with a constract containing the gene of interest under the control of the promoter containing the Gal4 binding elements and another construct expressing the engineered receptor, transcription of the gene of interest can be selectively controlled by administering the synthetic hormone

Intracellular receptors for steroid hormonesA peculiar type of steroid hormone receptor is the D. melanogaster ecdysone receptor (EcR). In insects, the steroid hormone ecdysone has an essential role in stimulating metamorphosis. The hormone acts by entering in the cells and binding a receptor composed of hetrodimer of two proteins, the EcR itself and the ultraspiracle (USP) gene productIntracellular receptors for steroid hormones In this manner, expression of the gene of interest only occurs after administration of ecdysone or its synthetic analog muristeron A or ponasteron A. In mammals, the pharmocokinetics of these hormones is very favorable, sincethey neither accumulate nor are eliminated too rapidly, are not toxic and are inactive in the absence of their cognate receptors. Furthermore, the lipophilic nature of these compounds allows them to freely cross the plasma membrane and the blood-brain barrier. Transcriptional control by ligand-induced transcription factor dimerizationA third system to achieve pharmacologically induced transcription is based on the property of the drug sirolimus (rapamycin, a macrolide antibiotic originally extracted from a Streptomyces)Transcriptional control by ligand-induced transcription factor dimerizationThis drug has been used for over a decade as an immunosuppressant in transplanted patients and is now also being tested for cancer therapy. Inside the cells, rapamycin interacts with a member of the FKBP (FK506-binding protein) family, the immunophilin FKBP12. The rapamycin/FKBP 12 complex then binds a specific domain (the FRB domain) of mTOR (mammalian target of rapamycin, a protein of the PBK family) protein kinase and blocks its action.

Transcriptional control by ligand-induced transcription factor dimerizationSince this kinase is essential for signal transduction in T cells, rapamycin causes cell cycle arrest in the Gl phase of the cell cycle; lack of T-cell proliferation explains the immunosuppressive effect of the drug. Rapamycin is thus the prototype of a drug selectively inducing dimerization of two proteins, FKBP12 and mTOR. Transcriptional control by ligand-induced transcription factor dimerizationWhen FKBP is fused to the zinc linger (ZF) domain of a transcription factor and its FRB partner to a transcriptional activation domain, the presence of rapamycin induces the formation of a heterodimeric transcription factor; this synthetic factor binds a specitic target sequence thanks to the ZF domain and activates transcription through its activation

transcription can be activated over 10,000-fold in the induced condition compared to the basal level. However, they require that, together with the therapeutic gene, the gene coding for the inducible regulator is also transferred into the same cell. In addition, the regulator often consists of two different proteins, as in the case of the ecdysone- or rapamycin-regulated systems. This poses important limitations to the clinical experimentation, since it obliges the experimenter to accommodate several transcriptional cassettes within the same vector, and to use vectors allowing cloning of large DNA inserts.ln addition, the regulatory proteins must be expressed in a constitutive manner; however, these proteins derive from different organisms and are thus potentially immunogenic. Finally, the above-described inducible systems can efficiently regulate RNA polymerase II transcription, while they are difficult to adapt to RNA polymerase lll regulation, which, by its own nature, tends to be constitutively active. Thus, the inducible systems are effective in regulating protein- and microRNA-coding genes, but much less the genes coding for shRNAs or other small regulatory RNAs

2.2 Non-Coding Nucleic AcidsBesides protein-coding nucleic acids, the spectrum or gene therapy applications is enormously increased by the possibility or using short nucleic acids (DNAs or RNAs) with regulatory function. These molecules belong to one of at least six possible classes: oligonucleotides and modified oligonucleotides; small catalytic RNAs and DNAs (ribozymes and DNAzymes) small regulatory RNAs (siRNAs and microRNAs); long antisense RNAs; decoy RNAs and DNAs; RNAs binding to other molecules thanks to their tridimensional structure (aptamers).

DNA oligonucleotides and modified oligonucleotides must be administered to the cells from the outside. In contrast, the RNA molecules can also be synthesized inside the cells by transferring their coding DNA sequences. all steps regulating gene expression can be controlled by small regulatory molecules, includingtranscription and splicing (oligonucleotides), mRNA decay (oligonucleotides, ribozymes, DNAzymcs, siRNAs and long antisense RNAs), protein synthesis (siRNAs), and protein function (aptamers and decoys).

2.2.1 OligonucleotidesThe simplest form of nucleic acids with potential therapeutic function consist in short, chemically synthesized, single-stranded DNA molecules, usually 15-100 nt long. Use of these oligodeoxynucleotides is based on the intrinsic property of a DNA strand to pair with its complementary sequence thanks to the formation of hydrogen bonds between the nucleotide bases. 2.2.1 OligonucleotidesAlthough each of these bonds is non-covalent and thus weak, their overall number renders the overall affinity between two complementary DNAFurthermore, base pairing is extremely specific in terms of target recognition, since a 17-nt DNA stretch is statistically present only once in the entire human genome. 2.2.1 Oligonucleotidessynthetic oligodeoxynucleotides can be used in at least four different types of applicalions: the first two targeting cellular mRNA and the second two genomic DNA; to block gene expression, exploiting base paring between the oligodeoxynucleotides and a target RNA sequence; to modulate pre-mRNA splicing, thus favoring or impeding inclusion of one exon in the mature mRNA;to block transcription, by promoting formation of triple helix DNA structures, usually in the promoter region of a target gene; to promote gene correction, exploiting oligodeoxynucleotide pairing with a homologous genomic DNA segment. Antisense oligodeoxynucleotides (ASOs) blocking gene expression To selectively inhibit expression of a cellular or viral gene, 17-22-nt oligodeoxynucleotides can be used, having a sequence complementary to that of the target mRNA. Once introduced into the cell, pairing of the ASO with its target blocks ribosomal translation and stimulates degradation of the RNA:DNA hybrid by cellular RNase H enzymes.Antisense oligonucleotides (ASOs) that modulate splicingThanks to its complementarity to target mRNA, an ASO can also be used to induce exclusion of an exon during premRNA maturation. ln some diseases, presence of a point mutation in an exon can generate a stop codon, leading to the synthesis of a prematurely truncated protein, or shifting the open rending frame downstream of the mutation. Antisense oligonucleotides (ASOs) that modulate splicingIn some situations, such as Duchenne muscular dystrophy (DMD), the disease caused by such mutations is much worse than that eventually induced by the absence or the entire exon containing the mutation. One strategy to induce exon skipping from an mRNA is to treat cells with an ASO complementary to the signals that regulate pre-mRNA splicing. These ASO typically target the 5' and 3' splice sites or the sequences internal to the exon that determine its retention in the mature mRNA (exon sequence enhancers, ESEs).

Triple helix forming oligodeoxynucleotides (TFOs)TFOs are single-stranded oligodeoxynucleotides binding the DNA major groove in a sequence-specific manner. In particular, they form very stable bonds with a DNA duplex of 10-30 bp, containing a stretch of purines and a complementary stretch of pyrimidines. The third helix is formed by the TFO, which, according to the target sequence, can be made of either purines or pyrimidines.

Triple helix forming oligodeoxynucleotides (TFOs)The TFO binds the target duplex through the formation of two hydrogen bonds between each of its bases and the purine-rich strand of the target DNA. Formation of a triple helix is thus an intrinsic property of DNA structure and does not require any cellular protein. Triple helix forming oligodeoxynucleotides (TFOs)When the TFO target is in the promoter of a gene, or immediately downstream of the transcription start site, the triple helix structure impair transcription factor binding or duplex DNA unwinding, thus blocking gene expression. Triple helix forming oligodeoxynucleotides (TFOs)In some instances, formation of a triple helix was also shown, although at low efficiency, to promote DNA repair, thus inducing the correction of mutations, by recruiting the cellular machineries responsible for excision repair or homologous recombination.

Oligonucleotides inducing correction of point mutationsOne of the most ambitious goals of gene therapy is to directly modify the genetic information to obtain the correction of a pathological mutation. Oligonucleotides inducing correction of point mutationsThis objective can be met by introducing, into the cells a DNA stretch having a sequence identical to the region to be corrected, however without the mutation, and then exploiting the cellular machinery involved in DNA repair for the substitution of the genomic DNA sequence with that administered exogenously. Oligonucleotides inducing correction of point mutationsgene conversion process is usually based on the cellular machinery responsible for homologous recombination, and uses, as substrates, long, single-stranded DNA stretches, composed of several thousand nucleotides. Oligonucleotides inducing correction of point mutationsIn some cases, however, short, single-stranded oligonucleotides, composed of 20-110 nt and having mismatched nucleotides in their central position, can stimulate gene correction by favoring the recruitment of cellular DNA mismatch repair protein. Although this approach is experimentally interesting, the fequency at which gene correction can be obtained is usually very low (usually less than O.l% of the treated cells) and still restricted to cultured cells.

2.2.2 Modified Oligonucleotides Among the essential problems limiting application of oligodeoxynuclcotides in vivo is their limited tissue distribution, cytotoxicity, andlow stability. 2.2.2 Modified Oligonucleotides Both in the extracellular environment and inside the cell, short natural DNA molecules, in which nucleotides are connected by phosphodiester bonds, are rapidly degraded by endo- and exonucleases (DNases). To overcome this problem and thus increase bioavailability of these molecules in vivo, the oligodeoxynucleotides structure can be altered by introducing various chemical modifications Modified Oligonucleotides first-generation In first-generation modified oligonucleotides, one non-bonding oxygen atom in the phosphate group is substituted by a sulfur atom (phosphorothioate oligodeoxynucleotides). This modification confers higher stability to the molecules and thus increases their half-life.

Modified Oligonucleotides first-generation In vitro, phosphorothioate oligodeoxynucleotides can be efficiently delivered to cells using lipofection.In vivo, they can be used in the form of naked molecules, however their half-life is very short (less than two hours in serum and four hours in tissues) and therefore they need to be administered by continuous intravenous infusion. Phosphorothioate oligodeoxynucleotides are relatively well tolerated, with minimal side effects probably due to the interaction of the polyanion backbone with some serum proteins. Modified Oligonucleotides second generation A second generation of modified oligonucleotides was obtained by the introduction of an alkyl group in the 2' position or the ribose molecule of nucleotides. The generated molecules include '2'-0-methy- and '2'-0-methoxyethyl-RNA. These molecular species are less toxic than phosphorothioate oligodeoxynucleotides.

Modified Oligonucleotidessecond generation The conformation changes induced by these modifications and, in particular, the presence of ribose, improve target binding, however they also abrogate the oligonucleotide:target mRNA duplex ability to activate RNase H, a crucial aspect of ASO activity. Thus, the inhibitory effect of these molecules is only exerted through the inhibition of mRNA translation and is thus lower than that of phosphorothioates. Modified Oligonucleotides second generation This drawback can be avoided by generating chimeric oligonucleotides with 2'-modified nucleotides placed only at the ends of the oligonucleotide, thereby leaving a central RNase-compatible DNA gap. These hybrid oligonucleotides are known as gapmers.