DNA-binding protein for targeted nucleic acids delivery

Abstract

Are disclosed peptide binding the CD71 transferring receptor, genetically engineered DNA-binding proteins from starved cells (DPS), and hybrid protein-nucleic acid complexes having the capability of being identified and taken up by the human CD71 receptor, and which can be used in targeted delivery of nucleic acid in cells.

Description

[0001] DNA-BINDING PROTEIN FOR TARGETED NUCLEIC ACIDS DELIVERY

[0002] Field of the invention

[0003] The present invention relates to the field of biotechnology and more in details to peptide binding the CD71 transferring receptor, genetically engineered DNA-binding proteins from starved cells (DPS), and hybrid protein-nucleic acids complexes having the capability of being identified and taken up by the human CD71 receptor, and which can be used in targeted delivery of nucleic acid in cells.

[0004] State of the art

[0005] Over the past two decades, significant technological innovations have led to mRNA becoming a promising option for developing prophylactic and therapeutic vaccines, protein replacement therapies, and genome engineering. RNA-based therapeutics have shown tremendous promise in disease intervention at the genetic level, and some have been approved for clinical use, including the recent CO VID-19 messenger RNA (mRNA) vaccines against the novel coronavirus (SARS-CoV-2) pneumonia epidemic. The success of the two COVID-19 mRNA vaccines has sparked new enthusiasm for other medical applications, particularly in cancer treatment. Messenger RNA has emerged as a new and efficient agent for the treatment of various diseases [Sun H, Zhang Y, Wang G, Yang W, Xu Y. mRNA-Based Therapeutics in Cancer Treatment. Pharmaceutics. 2023 Feb 13;15(2):622. doi: 10.3390 / pharmaceuticsl5020622. PMID: 36839944; PMCID: PMC9964383.]. The success of lipid nanoparticle-mRNA has proved the clinical potential of nanoparticle-mRNA formulations. Compared to other vaccines, the inherent properties of mRNA vaccines and their interaction with lipid nanoparticles (LNP) make them considerably unstable throughout their life cycles, impacting their effectiveness and global accessibility. An improvement of mRNA vaccine stability and investigation of the factors influencing stability will be imperative. Since mRNA structure, excipients, LNP delivery systems, and manufacturing processes are the primary factors affecting mRNA vaccine stability, optimizing mRNA structure and screening excipients can effectively improve mRNA vaccine stability [Cheng F, Wang Y, Bai Y, Liang Z, Mao Q, Liu D, Wu X, Xu

[0006] M. Research Advances on the Stability of mRNA Vaccines. Viruses. 2023 Mar 2;15(3):668. doi: 10.3390 / vl5030668. PMID: 36992377; PMCID: PMC10051489., 2023; Pesque D, Pujol RM, Marcantonio O, Vidal -Navarro A, Ramada JM, Arderiu-Formenti A, Albalat-Torres A, Serra C, Gimenez -Arnau AM. Study of Excipients in Delayed Skin Reactions to mRNA Vaccines: Positive Delayed Intradermal Reactions to Polyethylene Glycol Provide New Insights for COVID-19 Arm. Vaccines (Basel). 2022 Nov 30;10(12):2048. doi: 10.3390 / vaccinesl0122048. PMID: 36560458; PMCID: PMC9788122.]. However, the deficiency in the effective biological distribution, high transfection efficiency and good biosafety are still the major challenges in clinical translation of nanomedicine for mRNA delivery. The clinical success of RNA therapy is largely dependent on the use of chemical modification, ligand conjugation or non-viral nanoparticles to improve RNA stability and facilitate intracellular delivery in the developing safe and effective delivery systems. Moreover, improving manufacturing processes could also prepare thermally stable mRNA vaccines with safety and efficacy.

[0007] To date, a variety of promising nanoparticles have been constructed and then gradually optimized to facilitate the effective biodistribution of carriers and efficient nucleic acid delivery [Sharifnia Z, Bandehpour M, Hamishehkar H, Mosaffa N, Kazemi B, Zarghami

[0008] N. In-vitro Transcribed mRNA Delivery Using PLGA / PEI Nanoparticles into Human Monocyte-derived Dendritic Cells. Iran J Pharm Res. 2019 Fall; 18(4): 1659-1675. doi: 10.22037 / ijpr.2019.1100872. PMID: 32184837; PMCID: PMC7059071.19; Zhang, Dapeng, et al. "The unexpected importance of the primary structure of the hydrophobic part of one-component ionizable amphiphilic Janus dendrimers in targeted mRNA delivery activity." Journal of the American Chemical Society 144.11 (2022): 4746-4753; Chen et al., 2022; Chyi, Lim Chiou, Chia Le Yi, and Palanirajan Vijayaraj Kumar. "Dendrimer-based nanocomposites for the production of RNA delivery systems." OpenNano (2023): 100173; Kysakova, Lyubov A., et al. "Dendrimer-Mediated Delivery of DNA and RNA Vaccines." Pharmaceutics 15.4 (2023): 11063], Additionally, advanced strategies of mRNA delivery techniques such as viral delivery, self-assembled RNA- triple-helix hydrogel drug delivery systems, and hyaluronic acid / protamine sulfate inter- polyelectrolyte complexes have been reported [Lechner F, Jegerlehner A, Tissot AC, Maurer P, Sebbel P, Renner WA, Jennings GT, Bachmann MF. Virus-like particles as a modular system for novel vaccines. Arteriology. 2002;45(4-6):212-7. doi: 10.1159 / 000067912. PMID: 12566703; Li X, Guo X, Hu M, Cai R, Chen C. Optimal delivery strategies for nanoparticle-mediated mRNA delivery. J Mater Chem B. 2023 Mar 8;l l(10):2063-2077. doi: 10.1039 / d2tb02455a. PMID: 36794598],

[0009] Some examples of protein-based carriers derived from bacteria have been reported for their application in vaccine development to enhance the immune response in support of the feasibility of the employment of said nanoparticles in the field. The DNA-binding proteins from starved cells (DPS), are conserved in prokaryotic organism and in many species, function as a double strands DNA binding protein protecting nucleic acids in prokaryotic cells from oxidative damage by forming DPS-DNA or RNA complexes [Orban K, Finkel SE. DPS Is a Universally Conserved Dual-Action DNA-Binding and Ferritin Protein. J Bacteriol. 2022 May 17;204(5):e0003622. doi: 10.1128 / jb.00036-22. Epub 2022 Apr 5. PMID: 35380871; PMCID: PMC9112962.]. All DPS-like proteins are characterized by a common three-dimensional architecture and are found as spherical dodecamers with a hollow central cavity which is strikingly like ferritin protein structures [Haikarainen, Teemu, and Anastassios C. Papageorgiou. "DPS-like proteins: structural and functional insights into a versatile protein family." Cellular and molecular life sciences 67 (2010): 341-351], The existence of positively charged modules with functional significance implies a straightforward electrostatic interaction between DPS and nucleic acid. Studies have shown that DPS can bind to a variety of nucleic acids, including single-stranded DNA, double-stranded DNA, and RNA [Park C, Jin Y, Kim YJ, Jeong H, Seong BL. RNA-binding as chaperones of DNA binding proteins from starved cells. Biochem Biophys Res Commun. 2020 Apr 2;524(2):484-489. doi: 10.1016 / j .bbrc.2020.01.121. Epub 2020 Jan 30. PMID: 32007271; Dubrovin E.V., L A. Dadinova, M.V. Petoukhov, et al “Spatial organization of DPS and DNA-DPS complexes” Journal of Molecular Biology 433 (2021) 166930],

[0010] DNA-binding proteins from starved cells (DPS) constitute a family of highly conserved proteins present in bacteria and archaea but not in human. Since their expression is found to be upregulated in response to stress and starvation conditions in cells, these proteins are crucial in safeguarding cellular DNA against free radicals (Almiron et al., 1992, A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli, Genes Dev, Dec;6(12B):2646-54. doi: 10.1101 / gad.6.12b.2646). DPS forms a dodecameric nanoparticle well conserved among all DPS with an outer and inner diameter of 9 nm and 4.5 nm, respectively (Grant RA, Filman DJ, Finkel SE, Kolter R, Hogle JM. 1998. The crystal structure of DPS, a ferritin homolog that binds and protects DNA. Nat StructBiol 5:294-303. 10.1038 / nsb0498-294). DPS are composed of 12 identical subunits with a molecular weight of about 20 kDa. Each DPS subunit folds into a compact five- helices bundle inter-connected by four loop sequences of variable lengths. Crystal structures of DPS from several bacteria and archaea have been determined, including E. Coli (Almiron et al., 1992, A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli, Genes Dev, Dec;6(12B):2646-54. doi: 10.1101 / gad.6.12b.2646), Sulfolobus solfataricus (Gauss GH, Benas P, Wiedenheft B, Young M, Douglas T, Lawrence CM. Structure of the DPS-like protein from Sulfolobus solfataricus reveals a bacterioferritin-like dimetal binding site within a DPS-like dodecameric assembly. Biochemistry. 2006 Sep 12;45(36): 10815-27. doi: 10.1021 / bi060782u. PMID: 16953567; PMCID: PMC1815386.), Mycobacterium smegmatis (Roy, Siddhartha, et al. "Role of N and C-terminal tails in DNA binding and assembly in DPS: structural studies of Mycobacterium smegmatis DPS deletion mutants." Journal of molecular biology 370.4 (2007): 752-767.), Deinococcus radiodurans (Kim, Song-Gun, et al. "Crystal structure of DPS-1, a functionally distinct DPS protein from Deinococcus radiodurans." Journal of molecular biology 361.1 (2006): 105-114. Stillman et al., Mol Microbiol. 57:1101-12, 2005), and Listeria innocua (Ilari, Andrea, et al. "The dodecameric ferritin from Listeria innocua contains a novel intersubunit iron-binding site." Nature Structural Biology 7.1 (2000): 38-43. Zhao et al., J Biol Chem 277:27689- 27696, 2002). DPS protects DNA against the oxidative stress through iron binding and DNA binding (Haikarainen T, Papageorgiou AC. DPS-like proteins: structural and functional insights into a versatile protein family. Cell Mol Life Sci. 2010 Feb;67(3):341- 51. doi: 10.1007 / s00018-009-0168-2. Epub 2009 Oct 14. PMID: 19826764). At the interface between subunits, DPS display a Fe2+binding site to prevent generation of toxic hydroxyl radicals through Fenton reaction that damage DNA. Furthermore, the N- or C- termini of DPS in many members of DPS is rich in basic amino acids and involved in the DNA binding through ionic interaction providing the physical protection of DNA (Ceci, Pierpaolo, et al. "DNA condensation and self-aggregation of Escherichia coli DPS are coupled phenomena related to the properties of the N-terminus." Nucleic Acids Research 32.19 (2004): 5935-5944).

[0011] International patent application, publication n. WO2011082087A2 discloses DPS used to deliver DNA vaccines in mice and demonstrates its potential as a vaccine carrier; the recombinant DPS proteins were reported in fusion with peptides derived by viral proteins to induce antibody responses in the absence of adjuvants resulting in protection against viral infection and allergic reactions. However, these systems do not use DPS for mRNA delivery and do not include the insertion of a CD71 -binding peptide, which provides the vehicle with high targeting properties for example to cancer cells and dendritic cells where CD71 is over-expressed providing great advantages in the development of new and improved vaccines [Lippitsch A, Chukovetskyi Y, Baal N, Bein G, Hackstein H. Unique high and homogenous surface expression of the transferrin receptor CD71 on murine plasmacytoid dendritic cells in different tissues. Cell Immunol. 2017 Jun;316:41-52. doi: 10.1016 / j.cellimm.2017.03.005. Epub 2017 Mar 29. PMID: 28372797], CD71 is a transmembrane glycoprotein that plays a crucial role in cellular iron homeostasis. It is primarily involved in the uptake of transferrin-bound iron, a process essential for various cellular functions, including DNA synthesis, cell proliferation, and erythropoiesis. CD71 is expressed on the surface of many cell types, with particularly high levels in rapidly dividing cells such as those found in the bone marrow, developing erythrocytes, and certain cancer cells. Due to its involvement in cell growth and proliferation, CD71 has gained attention as a potential target for therapeutic interventions, including cancer treatments [Wang J, Tian S, Petros RA, Napier ME, Desimone JM. The complex role of multivalency in nanoparticles targeting the transferrin receptor for cancer therapies. J Am Chem Soc. 2010 Aug 18; 132(32): 11306-13. doi: 10.1021 / jal043177. PMID: 20698697; PMCID: PMC2923393, 2010; Shinde SS, Ahmed S, Malik JA, Hani U, Khanam A, Ashraf Bhat F, Ahmad Mir S, Ghazwani M, Wahab S, Haider N, Almehizia AA. Therapeutic Delivery of Tumor Suppressor miRNAs for Breast Cancer Treatment. Biology (Basel). 2023 Mar 19;12(3):467. doi: 10.3390 / biology 12030467. PMID: 36979159; PMCID: PMC 10045434], Strategies that leverage the high expression of CD71 on certain cancer cells aim to selectively deliver cytotoxic agents or nanoparticles for more effective and targeted therapies.

[0012] International patent application, publication n. WO2021 / 076546 discloses an immunoglobulin belonging to the family of fibronectin type III that can tolerate several mutations retaining its structure and able to recognize CD71.

[0013] US patent n. US7608268 discloses ferritin nanocaged-structure for delivering small molecules including therapeutics drug or diagnostic ingredients. Indeed, ferritins are natural nanocages with an 8.5 A diameter hollow made of 24 identical subunits able to be internalized by transferrin receptor in a region localized in the apical domain in contrast with transferrin that binds CD71 in the region formed by the protease-like and the helical domains. Each ferritin subunit is approximately a 20 kDa protein composed of a helix bundle which includes a four-antiparallel helix motif, with a fifth shorter helix lying roughly perpendicular to the long axis of the 4-helix bundle and a surface-exposed loop B-C loop connecting helix B and C.

[0014] The binding site for CD71 is locate on the heavy chain of Ferritin protein (Li L, Fang CJ, Ryan JC, et al. Binding and uptake of H-ferritin are mediated by human transferrin receptor-1. Proc Natl Acad Sci U S A. 2010;107(8):3505. doi: 10.1073 / PNAS.0913192107; Montemiglio LC, Testi C, Ceci P, et al. Cryo-EM structure of the human ferritin-transferrin receptor 1 complex. Nat Commun. 2019;10(l): 1-8. doi: 10.1038 / s41467-019-09098-w).

[0015] Closest prior art

[0016] De Turris V. et al, Humanized archaeal ferritin as a tool for cell targeted delivery, Nanoscale, 9(2) 2016 disclose human Ferritin - Archaeoglobus (A.) fulgidus ferritin fusion / chimera comprising the BC-loop peptide RGGRIFLQDIKKPDSEWES; the chimera being recognized by transferrin receptor 1. Moreover, discloses ferritin for drug delivery including nucleic acids, tumour cells overexpressing TfRl, cancer therapy and diagnosis, the BC-loop being the best candidate for TfRl receptor recognition of the ferritin molecule and immune response.

[0017] US patent n. US9241986 discloses DNA-binding protein from starved cells (Ops) fusion proteins with proteins or peptides inserted into an internal site of Ops in one or more of the three loop sequences or the four helixes, chimeric Ops proteins such as one having one or both terminal sequences from one Ops and the internal sequence from another Ops to ensuring that both N- and C-termini are properly extended on the surface, ferritin fusion peptides / proteins, cancer vaccines, Ops coming from bacteria commonly associated with humans and animals, such as members of Escherichia, Lactobacillus, and Bacteroides and extremophilic or hyperthermophilic bacteria or archaea such as Sulfolobus solfataricus and Deinococcus radiodurans. Montemiglio L. C. et al., Cryo-EM structure of the human ferritin-transferrin receptor 1 complex, Nature Communications, 10 (1) 2019 disclose the external BC-loop of human ferritin (H-Ft) being one of three regions responsible for CD71 binding, significant binding capability of the humanized A. fulgidus / erritin where the human H-Ft BC-loop had been transplanted, likely due to the presence of F81 and Q83 on the loop, and to R22 on the A helix, CD71 being highly expressed in the most common cancer cell types, further highlighting the interest for this receptor as a privileged target for the selective delivery of cytotoxic drugs coupled to ferritin.

[0018] Japanese patent n. JP2021132623 discloses a Escherichia (E.) coli DPS protein.

[0019] Technical problem

[0020] Cancer cells and dendritic cells, that play a crucial role in the initiation of adaptive immune responses and the regulation of immune tolerance, overexpress CD71 receptor.

[0021] The inventors of the present invention, identified small peptides, located in the heavy chain of human ferritin (Uniprot code P02794, SEQ ID NO. 1) being the moiety sufficient and necessary to bind CD71. human ferritin (Uniprot code P02794) SEQ ID NO. 1:

[0022] MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFA KYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALH LEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKM GAPESGLAEYLFDKHTLGDSDNES

[0023] The same inventors designed genetically engineered DNA-binding protein from starved cells (so called DPS), belonging to the ferritin family, featuring the recognition motif for the CD71 receptor. The present inventors found that genetically engineered DNA-binding protein from starved cells (DPS), exhibits a notable affinity for nucleic acids and can be used as a carrier for nucleic acids, therefore, to obtain a safe tool for mRNA delivery, designed a hybrid molecule comprising the DNA-binding protein and nucleic acids.

[0024] Said complexes, made of genetically engineered DNA-binding protein from starved cells (DPS), and nucleic acids, need to be capable of binding to a wide range of sequences, to deliver nucleic acids without targeting specific genes or sequences, since a gene specific delivery is not required. Therefore, they enhance the delivery of multiple, unspecified, and functional sequences of nucleic acids.

[0025] Another distinguishing feature is that in the complexes, nucleic acid wraps around the proteins so that the complexes assume, when folded, a nanocage tridimensional conformation. Said tridimensional structures protect nucleic acids from degrading enzymes, rendering it suitable for targeted delivery to both malignant and regulating immune system cells.

[0026] The nanocages may overcome the traditional issues that emerge from inefficiency of m- RNA delivery, toxicity, or side effects of viral- and nonviral vehicles displaying low cytotoxicity and an excellent cell viability as well.

[0027] Moreover, the delivery of mRNA by means of genetically engineered DNA-binding protein from starved cells (DPS), is a rapid and simple process that does not require the production of viral vectors or the use of complex transfection protocols. This makes it a cost-effective and time-efficient method for gene expression studies or therapeutic applications.

[0028] Differently from prior art, the distinguishing feature of the present invention is the use of CD71 -binding peptide inserted in DPS proteins, wherein said peptides have a purposive sequence and may have specific mutations. Said characteristics Allow the use of the invention for dual targeting: oncological and immunotherapeutic.

[0029] In particular De Turris V. et al, 2016 does not disclose or suggest the use of nucleic acids and does not describe a ferritin and nucleic acid complex, while the present invention shows functional advantages, such as efficient internalization and / or selective immune activation.

[0030] Thus, the objects of the present invention can be used in oncology, immunotherapy, gene therapy, thanks to their unique molecular structures, by functional selectivity towards CD71 and by their effect of internalization and activation of the immune response, which is neither predictable nor suggested by the known technique. On the contrary, the effects of the construct disclosed in De Turris V. et al, 2016 are due to the intrinsic immunogenicity of said chimeric construct.

[0031] Even if the closest prior art suggests the preparation of protein-nucleic acid complexes, does not give hint to combine CD71 with nucleic acids, in particular with a length higher than 80 bp.

[0032] In summary, the closest prior art does not suggest a complex comprising a DPS protein containing a unique CD71 binding peptide with specific sequence and mutations. The object of the present invention is not mere optimization but derive from a new and non- obvious combination of genetic engineering to obtain a selective internalization via CD71.

[0033] OBJECT OF THE INVENTION

[0034] The above technical problem is solved by providing:

[0035] Peptide binding the CD71 transferring receptor, having a length of at least 12 amino acids, having a sequence being any sequence from position 77 to position 94 of human ferritin of SEQ.ID.NO 1; genetically engineered DNA-binding proteins from starved cells (DPS), comprising a CD71 binding peptide as above defined; hybrid complex of a genetically engineered DNA-binding protein from starved cells (DPS) comprising a CD71 binding peptide and at least one nucleic acid; the hybrid complex of a genetically engineered DNA-binding protein from starved cells (DPS) comprising a CD71 binding peptide and at least one nucleic acid for use as a carrier of nucleic acids in CD71 expressing cells, for use for internalization of nucleic acids in CD71 expressing cells , for use in gene therapy of CD71 expressing cells, for use for triggering innate immune response in CD71 expressing immune cells, for use for the treatment of cancer, for use in the treatment of pathogens infections, pharmaceutical composition thereof.

[0036] Further features of the present invention would be clear from the following detailed description with reference to the claims, the attached drawings and the experimental data provided.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 shows the production and expression of chimeric DPS-Ferritin (DPS-F) analyzed by SDS-PAGE. A) The gel displays: Ml) Protein marker, PCI) protein control (2pg), PC2) protein control (2pg); NC) Cell lysate without induction; 1) Cell lysate with induction for 16h at 15°C; 2) Cell lysate with induction for 4 h at 37°C; NCI) Supernatant of cell lysate without induction; 3) Supernatant of cell lysate with induction for 16h at 15°C; 4) Supernatant of cell lysate with induction for 4 h at 37°C; NC2) Pellet of cell lysate without induction; 5) Pellet of cell lysate with induction for 16 h at 15 °C; 6) Pellet of cell lysate with induction for 4 h at 37°C. B) Western blot displays M2) Western blot marker 3) Supernatant of cell lysate with induction for 16h at 15°C; 4) Supernatant of cell lysate with induction for 4 h at 37°C.

[0039] Figure 2 shows the characterization of chimeric DPS-Ferritin (DPS-F) A) Purification analyzed by SDS-PAGE: Lane 1 displays sample eluted from HisTrap column at 500 mM imidazole; lane 2 showed DPS-F protein eluted from gel filtration S400 column in 20 mM Tris, pH 7.4, 300 mM NaCl. B) Circular dichroism of 0.5 mg / mL DPS-F in 10 mM Tris pH 7.4, 100 mM NaCl.

[0040] Figure 3 shows the uptake of 300 nM Rod-DPS-F by MEG01 cells after 3 h treatment. Flow cytometer analysis. The gate indicates the percentage of Rhodamine-positive cells (99.7). For each sample 10,000 events gated on live cells were acquired.

[0041] Figure 4 shows a schematic view of the structure of GFP, FLAG-tagged SNRNP70 mRNA.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] The CD71 binding peptide of at least 12 amino acids, derives from the heavy chain of human ferritin residues from 77 to 94 of SEQ.ID.NO 1.

[0044] CD71 binding peptide-14 is SEQ ID NO 2: RGGRIFLQDIKKPD

[0045] The CD71 binding peptide may include an amino acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or homology to SEQ. ID NO 2

[0046] The genetically engineered DNA-binding proteins from starved cells (DPS), may comprise CD71 binding peptide as above defined.

[0047] DPS of E. Coli is of sequence SEQ ID NO. 3 MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0048] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQLGGVALGTTQVINSKTPLKS

[0049] YPLDIHNVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDKFL WFIESNIE wherein CD71 binding peptide is inserted therein.

[0050] Preferably, the genetically engineered DNA-binding protein from starved cells (DPS) comprising the CD71 binding peptide is selected from the group consisting of:

[0051] SEQ.ID.NO. 4 is SEQ ID NO 3 wherein SEQ ID NO. 2 is inserted in position 87 and length of DPS is unaltered (173 amino acids)

[0052] SEQ ID NO 4 (called DPS-F1):

[0053] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0054] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRIFLQDIKKPGTQVINSK

[0055] TPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAASRDL DKFLWFIECNIE

[0056] SEQ.ID.NO. 5 is SEQ ID NO. 3 wherein SEQ ID NO 2 is inserted in position 87 and a corresponding region of SEQ ID NO 2 is deleted to maintain a length of 162 amino acids

[0057] SEQ ID NO 5 (called DPS-F2):

[0058] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0059] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRAFLGTQDIKKPDLKSY

[0060] PLDIHNVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDKFLW FIECNIE SEQ.ID.NO. 6 is SEQ ID NO. 3 wherein SEQ ID NO 2 is inserted in position 87 and SEQ. ID NO. 3 is mutated in the region 87-110 and have length of 166 amino acids

[0061] SEQ ID NO 6 (called DPS-F3):

[0062] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0063] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRAFLGTQDIKKPDLKSY

[0064] PLDIHNVQDHLEELADRYAIVANDVREAIGEAEDDDTADILTAASRDLDKFLWF IECNIE

[0065] Preferably, SEQ ID NO 4 may further comprise at least one mutation K to E.

[0066] Preferably SEQ ID NO 5-6 may further comprise at least one mutation S to C.

[0067] Preferably SEQ ID NO 5-6 may further comprise at least one mutation K to E.

[0068] Preferably the K to E is in position 118 and or 132 and / or 139 wherein the numbering refers to SEQ. ID NO. 3

[0069] In a preferred embodiment mutated SEQ ID NO 6 is selected from the group consisting of:

[0070] SEQ ID NO 7 (being SEQ ID NO 6 with a mutation K to E in position 118, called

[0071] DPS-F3 KE 118):

[0072] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0073] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRAFLGTQDIKKPDLKSY

[0074] PLDIHNVQDHLEELADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDKFLWF IECNIE

[0075] SEQ. ID NO 8 (being SEQ ID NO 6 with a mutation K to E in position 132, called DPS- F3 KE 132): MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0076] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRAFLGTQDIKKPDLKSY

[0077] PLDIHNVQDHLKELADRYAIVANDVREAIGEAKDDDTADILTAASRDLDKFLWF IECNIE

[0078] SEQ. ID NO 9 (being SEQ ID NO 6 with a mutation K to E in position 139 called DPS- F3 KE 139):

[0079] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0080] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRAFLGTQDIKKPDLKSY

[0081] PLDIHNVQDHLKELADRYAIVANDVRKAIGEAEDDDTADILTAASRDLDKFLWF IECNIE

[0082] SEQ. ID NO 10 (being SEQ ID NO 6 with a mutation K to E in position 118 and 132, called DPS-F3 KE 118 132):

[0083] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0084] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRAFLGTQDIKKPDLKSY

[0085] PLDIHNVQDHLEELADRYAIVANDVREAIGEAKDDDTADILTAASRDLDKFLWF IECNIE

[0086] SEQ. ID NO 11 (being SEQ ID NO 6 with a mutation K to E in position 118 and 139, called DPS-F3 KE 118 139):

[0087] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN

[0088] MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRAFLGTQDIKKPDLKSY

[0089] PLDIHNVQDHLEELADRYAIVANDVRKAIGEAEDDDTADILTAASRDLDKFLWF IECNIE

[0090] SEQ ID NO 12 (being SEQ ID NO 6 with mutation K to E in positions 118,132 and 139, called DPS-F3 EEE) MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWN MRGANFIAVHEMLDGFRTALIDHLDTMAERAVQRGGRAFLGTQDIKKPDLKSY PLDIHNVQDHLEELADRYAIVANDVREAIGEAEDDDTADILTAASRDLDKFLWF IECNIE

[0091] The genetically engineered DNA-binding protein from starved cells (DPS) may include an amino acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12.

[0092] The hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: i. a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and ii. at least one nucleic acid.

[0093] Preferably the nucleic acid in ii) has a length of more than 80 base pairs.

[0094] Said feature gives molecular stability and biological efficacy to the hybrid complex.

[0095] It is another object of the present invention a pharmaceutical composition comprising: at least one CD71 binding peptide selected from the group consisting of SEQ ID NO 2, any amino acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or homology to SEQ. ID NO 2, and / or at least one genetically engineered DNA-binding protein from starved cells (DPS) comprising a CD71 binding peptide selected form the group consisting of: SEQ ID NO 4-12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and / or at least one hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and at least one nucleic acid, and pharmaceutically acceptable excipients carrier, vehicle or diluent.

[0096] Preferably the pharmaceutical composition is a vaccine.

[0097] The pharmaceutically acceptable excipients will be chosen depending on the form and administration and dosage by the person expert in the field based on is common general knowledge.

[0098] The hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: i. a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and ii. at least one nucleic acid. can be used as a carrier for internalizing nucleic acids in cells expressing the CD71 receptor on the cell surface.

[0099] Cell expressing the CD71 receptor can be immune cells, cancer cells. The hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: i. a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and ii. at least one nucleic acid can be used in gene therapy to internalizing nucleic acids in cells expressing the CD71 receptor on the cell surface in order to produce a therapeutic effect through by altering gene expression of target genes.

[0100] The hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: i. a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and ii. at least one nucleic acid, can be used for triggering innate immune response in CD71 expressing immune cells, preferably dendritic cells.

[0101] Dendritic cells that are among the first immune cells to detect and respond to pathogens, initiating the innate immune response.

[0102] The pharmaceutical composition comprising: at least one CD71 binding peptide CD71 binding peptide- 14 selected from the group consisting of SEQ ID NO 2, any amino acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or homology to SEQ. ID NO 2, and / or at least one genetically engineered DNA-binding protein from starved cells (DPS) comprising a CD71 binding peptide selected form the group consisting of: SEQ ID NO 4-12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and / or at least one hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and at least one nucleic acid, and pharmaceutically acceptable excipients carrier, vehicle or diluent, can be used for the treatment and / or prevention of cancer and pathogens infections.

[0103] Preferably, cancer is selected from the group consisting of: liver cancer, uterine cancer, kidney cancer, pancreatic cancer, lung cancer, ovarian cancer, bone, and soft tissue tumors, as well as lymphatic and myeloid leukemia.

[0104] It is also disclosed a method for the internalization of nucleic acids by means of a hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: i. a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and ii. at least one nucleic acid, to be internalized in cells expressing the CD71 receptor on the cell surface by incubating the cells in the presence of said hybrid complex.

[0105] It is also disclosed a method for altering gene expression of target genes by administering to the patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising at least one CD71 binding peptide selected from the group consisting of SEQ ID NO 2, any amino acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or homology to SEQ. ID NO 2, and / or at least one genetically engineered DNA-binding protein from starved cells (DPS) comprising a CD71 binding peptide selected form the group consisting of: SEQ ID NO 4-12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and / or at least one hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and at least one nucleic acid, and pharmaceutically acceptable excipients carrier, vehicle or diluent.

[0106] It is also disclosed a method for triggering innate immune response in CD71 expressing immune cells, preferably dendritic cells by administering to the patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising at least one CD71 binding peptide CD71 selected from the group consisting of SEQ ID NO 2, any amino acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or homology to SEQ. ID NO 2, and / or at least one genetically engineered DNA-binding protein from starved cells (DPS) comprising a CD71 binding peptide selected form the group consisting of: SEQ ID NO 4-12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and / or at least one hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: a CD71 binding peptide as above disclosed selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and at least one nucleic acid, and pharmaceutically acceptable excipients carrier, vehicle or diluent.

[0107] It is also disclosed a method for the treatment of cancer and pathogens infections by administering to the patient in need thereof a therapeutically effective amount of a pharmaceutical composition at least one CD71 binding peptide selected from the group consisting of SEQ ID NO 2, any amino acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or homology to SEQ. ID NO 2, and / or at least one genetically engineered DNA-binding protein from starved cells (DPS) comprising a CD71 binding peptide selected form the group consisting of: SEQ ID NO 4-12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and / or at least one hybrid complex is made of a genetically engineered DNA-binding protein from starved cells (DPS) comprising: a CD71 binding peptide selected from the group consisting of: SEQ. ID NO 4 to 12, any acid sequence with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12, and at least one nucleic acid, and pharmaceutically acceptable excipients carrier, vehicle or diluent.

[0108] EXAMPLES

[0109] Example 1

[0110] Design of the genetically engineered protein DPS-Ferritin (DPS-F): DPS form E. coli has been modified by genetic engineering for being targeted and uptaken by cancer cells, by insertion in the region of the loop-alfa 3-loop of DPS from E. coli, residues 86 to 111, with the typical human H-homopolymer recognition by the transferrin receptor TfRl (or CD71) described as CD71 binding peptide.

[0111] Production of the chimeric protein DPS-Ferritin (DPS-F): the gene encoding for bacterial DPS-F was cloned into the expression vector pET28a (Novagen). The recombinant plasmid was transformed into E. coli TOP 10 cells and transformed into BL21(DE3)-Gold competent cells E. coli strain for protein expression (Agilent). For each construct, protein over-expression was obtained as follows: 1 L LB broth medium was inoculated with 2 ml overnight culture of a single colony and the gene expression was induced with 0.5 mM IPTG when the absorbance at 600 nm reached 0.6-0.8. Cells were harvested by centrifugation after overnight induction for 16h at 15°C (Lanes 1, 3 and 5) or at 4h at 37 °C (Lanes 2, 4 and 6) and the cell pellets were stored at -20°. Additionally, western blot gel was carried out to monitor and confirm the expression of the his-tagged protein.

[0112] Purification and characterization of the chimeric protein DPS-Ferritin (DPS-F): the purification method comprises the extraction from harvested cells in 40 ml buffer A (50 mM Tris / HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, and 5 mM MgCh) containing a cOmplete™ Mini Protease Inhibitor Cocktail Tablet (Roche) and disrupted by sonication. As reported in Fig. 1, the proteins were found to be expressed in the inclusion bodies hence, the pellets containing inclusion bodies were cyclically washed with buffer A added with 2% Tween 20 (Sigma- Aldrich) and centrifuged at 10000 RPM and the supernatant was removed. Water washing of the residual pellet was employed to remove the excess detergent. The pellets were then dissolved in 50 mL of buffer B consisting of 6 M urea, 50 mM Tris / HCl pH 7.5, 300 mM NaCl, and 1 mM TCEP and gently stirred overnight at 4 °C. The resulting solution was filtered with a 0.45 pm syringe filter and loaded onto a Nickel Sepharose High Performance affinity resin (HisTrap HP column, Cytiva). The column was washed with 5-7 column volumes of buffer B without TCEP to remove all unbounded proteins (flow through fraction in Fig.2). The protein was purified by steps of increasing imidazole and eluted at 250 mM imidazole. SDS-PAGE showed the presence of the pure His-tagged DPS-F characterized by a molecular mass of 19340 Da. The sample containing purified DPS-F protein was then dialyzed against 50 mM Tris pH 7.5, and 300 mM NaCl, to remove imidazole and Urea and promote the refolding. As a final purification step, the protein was concentrated to 2 mg / mL and loaded onto a HiLoad 26 / 600 S400 column by using an AKTA-Pure system (Cytiva) to assess the biological conformation and state of association of the mutated protein. Fractions containing highly purified protein were pooled, sterile filtered, and stored at 4 °C (Figure 2a). Protein concentration was calculated by measuring the UV spectrum using an extinction coefficient of 19480 M 'em1as a monomer. The purification process gives a yield of 20 mg of protein per liter of bacterial culture. Biological conformation was assessed by circular dichroism spectra confirming a high content of a-helices in agreement with 3D- structure (Figure 2b) (Grant RA, Filman DJ, Finkel SE, Kolter R, Hogle JM. 1998. The crystal structure of Dps, a ferritin homolog that binds and protects DNA. Nat Struct Biol 5:294-303. 10.1038 / nsb0498-294).

[0113] Cell cultures and internalization of chimeric protein DPS-Ferritin (DPS-F): to validate the ability of chimeric DPS-F to be up taken by cancer cells overexpressing TfRl receptor we used MEG01 cells that have been already assessed to over express TfRl receptor. MEG01 cells were purchase from ATCC (Manassas, VA, USA) and grown in RPMI-1640 medium supplemented with 100 lU / mL penicillin-streptomycin (P4458; Sigma-Aldrich), and 10% fetal bovine serum (F7524; Sigma-Aldrich) and kept inside a humidified incubator at 37 °C with 5% CO2. After trypsinization, MEG-01 cells were plated in a 96-multiwell plate and incubated with 300 and 600 nM of DPS-F labelled with NHS-Rodamine (DPS-Rod). MEG01 cells were washed two times with an imaging medium (DMEM without phenol red, 10% FBS, 10 mM Hepes, Glutamax and penicillin-streptomycin solution) to eliminate the unbound rhodamine-nanoparticles. For flow cytometry analysis, after incubation with Rod-DPS-F protein, MEG were washed two times with PBS, detached with Trypsin-EDTA (Euroclone, Milan, Italy), washed with Phosphate-Buffered Solution (PBS), and resuspended in BD-FACS Flow buffer. Control cells were treated in the same way but without Rod-DPS-F incubation. Internalization of conjugated nanoparticles before and after TB treatments was measured at the BD LSRFortessa (BD Biosciences, San Jose, CA, USA) and FACSDiva software (BD Biosciences version 6.1.3). Live cells were first gated using forward and side scatter area (FSC-A and SSC-A) plots, then detected in the red channel for Rhodamine signal (555 nm filter) and side scatter parameters. The gate for the final detection was set in the control sample. Data were analyzed using FlowJo9.3.4 software (Tree Star, Ashland, OR, USA). Both fluorescence microscopy and flow cytometry analysis confirmed an excellent internalization and localization into the cytoplasm after 3 hours treatment as shown in Fig 3.

[0114] Preparation of DPS-F-mRNA complex and assessment of the delivery of the complex into cancer cells: the nucleic acid-binding activity of DPS-F was assessed by using GFP, FLAG-tagged SNRNP70 mRNA (cod. 03-903 Merck) whose structure is shown in figure 4. The GFP, FLAG-tagged SNRNP70 mRNA is known to express green fluorescent protein (GFP) 24 h after conventional transfection in HeLa cells with a 77.5% of positive cells analyzed by flow cytometry.

[0115] DPS-F in 20 mM Tris, pH 7.5 and 150 mM NaCl (2 mg / mL) was added with m-RNA (ratio 1 : 1 mRNA / protein), previously resuspended in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0), and gentle mixed for 2 hours at room temperature. MEG-01 cells were grown as described previously and incubated with 300 DPS-F-mRNA complex for 24 hours. The expression of GFP, demonstrated the internalization of the mRNA through DPS-F nanocarrier and confirm the functionality of mRNA which is correctly translated, evidenced by bright field microscopic images of live MEG01 cells after 24 h of treatment with 300 nM DPS-F-mRNA (1 : 1), fluorescence stained with Hoechst 33342 evidencing also single channel of the GFP and the merge channels.

[0116] The proof of concept of the novel system composed of DPS-F to safely deliver a functional GFP-FLAG-tagged mRNA has been demonstrated by fluorescence microscope experiments. It has been shown that DPS-F is capable of entering target cells (MEG01) using the CD71 receptor, highly expressed in these cells and safely deliver mRNA into targeted cells. Furthermore, the DPS-F-mRNA nanocage system is able to deliver mRNA capable to correctly translate GFP expression as given from fluorescence microscope experiment.

Claims

CLAIMS1) A CD71 binding peptide of 12-18 amino acids, being any sequence from position 77 to position 94 of SEQ.ID.NO 1.2) The CD71 binding peptide-14 according to claim 1 of SEQ ID NO 2.3) The CD71 binding peptide according to claim 2 with at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or homology to SEQ. ID NO 24) Genetically engineered DNA-binding protein from starved cells (DPS) comprising a CD71 binding peptide of anyone of claims 1-3.5) Genetically engineered DNA-binding protein from starved cells (DPS) according to claim 4 is selected from the group consisting of: from SEQ.ID.NO. 4 to SEQ ID NO 66) Genetically engineered DNA-binding protein from starved cells (DPS) according to claim 5 wherein SEQ ID NO 4 comprising at least one mutation K to E.7) Genetically engineered DNA-binding protein from starved cells (DPS) according to claim 5 wherein SEQ ID NO 5 and / or SEQ ID NO. 6 comprising at least one mutation S to C.8) Genetically engineered DNA-binding protein from starved cells (DPS) according to claim 5 wherein SEQ ID NO 5 and / or SEQ ID NO. 6 comprising at least one mutation K to E.9) Genetically engineered DNA-binding protein from starved cells (DPS) according to claim 8 wherein K to E mutation is in position 118 and or 132 and / or 139 with numbering referring to SEQ. ID NO. 310) Genetically engineered DNA-binding protein from starved cells (DPS) according to claim 6 selected from the group consisting of from SEQ ID NO 7 to SEQ ID NO 1211) Genetically engineered DNA-binding protein from starved cells (DPS) according to anyone of claims 5-10 having at least 65%, 70%, 75%, 80%, 82 %, 85 %, 90 %, 92 %, 95%, 98%, 99% or 100% identity or similarity to SEQ. ID NO 4 to 12.12) A hybrid complex consisting of at least one genetically engineered DNA-binding protein according to anyone of claims 4-11 and at least one nucleic acid.13) The hybrid complex according to claim 12 for use as a carrier for internalizing nucleic acids in cells expressing the CD71 receptor on the cell surface.14) The hybrid complex according to claim 12 for use in gene therapy to internalizing nucleic acids in cells expressing the CD71 receptor on the cell surface in order to produce a therapeutic effect through by altering gene expression of target genes.15) The hybrid complex according to claim 12 for use for triggering innate immune response in CD71 expressing immune cells, preferably dendritic cells16) Use according to claim 13 wherein the cells are immune cells, cancer cells.17) A pharmaceutical composition comprising at least one CD71 binding peptide of anyone of claims 1-3 and / or at least one genetically engineered DNA-binding protein of anyone of claims 4-11 and / or at least one hybrid complex of claim 12 and pharmaceutically acceptable excipients carrier, vehicle or diluent.18) The pharmaceutical composition of claim 17 being in the form of a vaccine.19) The pharmaceutical composition according to anyone of claims 17-18 for use for treatment and / or prevention of cancer and pathogens infections.20) Use according to claim 19 wherein cancer is selected from the group consisting of: liver cancer, uterine cancer, kidney cancer, pancreatic cancer, lung cancer, ovarian cancer, bone, and soft tissue tumors, as well as lymphatic and myeloid leukemia.21) A method for internalizing nucleic acids in cells expressing the CD71 receptor on the cell surface by incubating said cells with a hybrid complex according to anyone of claims 12-15.

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