This page contains a Flash digital edition of a book.
Therapeutics


followed by reprogramming of acinar cells to insulin producing  cells.


Modality


The reprogramming factors can be proteins, small molecules, RNAs (including siRNAs, miRNAs and mRNAs) or DNAs (using virus or plasmid as vectors). From a practical standpoint, the DNA approach or gene therapy may have the most dif- ficulties in reaching the clinics, as shown by the tortuous history in the gene therapy field. However, the DNA approach is very useful in screening and selecting the right combination of reprogramming factors in the research and discov- ery stage, as demonstrated in several studies2-5. The strengths and limitations of the other modali- ties – small molecules, RNAs and proteins – are outlined in Table 4.


Certain small molecule drugs can exert the reprogramming effects by changing the epigenetic landscape, affecting signal transduction, or activat- ing/suppressing transcription factors. But finding the right small molecule drugs is not straightfor- ward – it requires extensive screening and optimi- sation. An advantage of small molecule drugs is that they are easy to synthesise, transport and administer, thus the costs of manufacture, distribu- tion and administration are low. However, like all other small molecule drugs, there are specificity issues: they can act on multiple types of cells, have off-target effects, or distribute all over the body. The development of RNA reprogramming agents is straightforward. Once the reprogram- ming factors are identified, the respective RNAs can be generated and evaluated8. But the biggest problem for the RNA approach is delivery: barri- ers to effective RNA delivery include low stability, low specificity and high toxicity upon systemic administration. Several years ago, siRNA was the hottest therapeutic option in the pipeline of the biopharmaceutical industry. But major pharmas’ excitement over RNA interference has faded due to the delivery problem. A recent example came last November when Roche announced it would end its in-house RNAi research.


Protein-based treatment may be more practical than RNA-based therapeutics. But to make it work, two hurdles in drug delivery have to be over- come. First, the reprogramming factors are usually transcription factors and chromosome remodelling proteins – they have to enter the nuclei to be func- tional. But in their native forms these proteins can- not be delivered into the cells. To overcome this obstacle, these proteins can be fused with protein transduction domains (PTDs). As a result, the engi-


Drug Discovery World Summer 2011


neered proteins are transducible, ie, capable of penetrating cells and trafficking into nuclei when placed in cell culture medium or around the affect- ed organs9. Secondly, most PTDs are not selective among different cell types. Ideally, the reprogram- ming proteins will only enter the substrate cells upon administration. To achieve this end, proteins can be engineered to target specific cell types. One strategy is to make the PTD with high affinity for certain cell types. A cell-specific PTD can be select- ed by screening a peptide library based on the phage display technology10. Another way is to construct a fusion protein in which the trans- ducible component is an antibody against a cell- specific surface receptor11. The fusion protein can enter the cells that specifically express the receptor through endocytosis.


Protein-based IVR can be very specific in terms of mechanism, location and cell type. Its draw- backs are the same as other protein drugs: the manufacturing and distribution cost is relatively high, compared to small molecule and RNA agents; and unlike small molecule drugs, they can not be taken orally.


The modality of IVR therapeutics is not exclu-


sive – for example, proteins and small molecules may be used in conjunction to achieve a higher reprogramming efficiency than that achieved by proteins alone12.


Protein-based IVR: example


In April 2009, a collaboration between our team and The Scripps Research Institute resulted in a major breakthrough in stem cell research12. For the first time mouse fibroblast cells were converted to iPSCs without the use of any exogenous genetic material. We engineered and produced four tran- scription factor proteins – oct4, sox2, klf4 and c- myc – that are capable of penetrating into and reprogramming cells. By using recombinant pro- teins we eliminated any risk of modifying the tar- get cell genome, making it possible to use iPSC and its derivative cells in a clinical setting. More impor- tantly, the study established the feasibility of using protein-based IVR in clinical settings. One application of the IVR technology is in the treatment of diabetes. Our strategy is to repro- gramme liver and/or pancreatic exocrine cells to insulin-producing -cells using the combination of transducible proteins Pdx1-PTD, Ngn3-PTD and MafA-PTD. Some preliminary data are pre- sented below.


Six CD-1 mice were divided into two groups: the treatment group and the control group. Transducible protein Pdx1-PTD (1mg/kg), Ngn3-


87


Continued from page 85


10 Brown, KC. New approaches for cell-specific targeting: identification of cell- selective peptides from combinatorial libraries. Current Opinion in Chemical Biology 4, 16-21 (2000). 11 Song, E, Zhu, P, Lee, S-K, Chowdhury, D, Kussman, S, Dykxhoorn, DM, Feng, Y, Palliser, D, Weiner, DB, Shankar, P, Marasco, WA, Lieberman, J. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nature Biotechnology 23, 709-717 (2005). 12 Zhou, H, Wu, S, Joo, JY, Zhu, S, Han, DW, Lin, T, Trauger, S, Bien, G, Yao, S, Zhu, Y, Siuzdak, G, Schöler, HR, Duan, L and Ding, S. Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins. Cell Stem Cell 4, 581 (2009). 13 Ber, I, Shternhall, K, Perl, S, Ohanuna, Z, Goldberg, I, Barshack, I, Benvenisti-Zarum, L, Meivar-Levy, I and Ferber, S. Functional, Persistent and extended liver to pancreas transdifferentiation. The Journal of Biological Chemistry 278, 31950-31957 (2003).


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92