Abstract:
The present invention provides for a process for transferring biomolecules such as polynucleotides and protein from cell to cell, eventually resulting in the transport of a biomolecular cargo throughout the entirety of one or more of a cell culture, tissue, organ, organ system, or organism.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    In the subject area of transfection for research, experimental, medical, and diagnostic purposes, viral vectors have traditionally been limited by immune reactions to the mechanism of DNA transfer, while non-viral vectors have traditionally been limited in their effectiveness and proliferation. 
         [0002]    Exosomes are intercellular vesicles created by the fusion of a multivesicular body, a vesicle containing one or more intra lumenal vesicles (ILVs), with the plasma membrane of a cell. Their lipid, protein, and RNA compositions differ substantially from those of their donor cells, indicating that there exist selective uptake mechanisms for transport into the ILVs that later become exosomes. Candidate factors for ILV translocation include lipids such as ceramide and proteins such as the ESCRT complexes, among other biomolecules. 
       SUMMARY OF THE INVENTION 
       [0003]    The present invention describes a novel process that results in the transfer of biomolecules such as polynucleotides and protein from cell to cell, eventually resulting in the transport of a biomolecular cargo throughout the entirety of one or more of a cell culture, tissue, organ, organ system, or organism. 
         [0004]    A Self-Iterating Factor (SIF) is a DNA-binding fusion protein that has an affinity for factors involved in ILV translocation such that the DNA attached to the SIF is incorporated into an ILV, which later becomes an exosome. According to the present invention, an exosome then fuses with a target cell, releasing the genetic construct that can be defined by one of many natural bioprocesses and then placed in the SIF, which serves as a robust transport method for biomolecular transfer such that desired bioprocesses can occur in the target biological system. These processes involve the alteration of one or more of: the genome, the transcriptome, and the proteome. 
         [0005]    Using its species-matched origin of replication, the genetic construct replicates before the SIF-bound copy of the construct is incorporated into yet another ILV, restarting the self-iterating cycle of transfection with the genetic construct. Specifically, the present invention teaches that the genetic construct codes for the SIF (among other gene products), such that new copies of the SIF-construct complex can be regenerated in the cytoplasm as time passes. Because the genetic construct is DNA shuttled between cells via an exosome, this DNA is known as exosomal shuttle DNA (esDNA). Unlike previously known processes, the process taught by the present invention does not repeat indefinitely, as the gene encoding the SIF contains signals for methylation or other nucleotide alterations that will decrease gene expression over time. 
         [0006]    A self-iterating exosomal vector therefore combines the proliferation of a viral vector with the immune non-recognition found in non-viral vectors due to the fact that the exosomes are derived from the target organism. 
         [0007]    The essence of the invention is that exosomes, intercellular transport vesicles, can provide a self-iterating transfer method for sending biomolecules such as nucleic acids and protein from cell to cell, particularly DNA. This culminates in the transfer of the biomolecular cargo throughout the entirety of a cell culture, tissue, organ, organ system, or organism. 
         [0008]    Exosomes arise from endosomal patches of the plasma membrane budding outward as well as from intralumenal vesicles of multivesicular bodies being released when the outer membranes of multivesicular bodies fuse with the plasma membrane. A genetic construct encoding a DNA-binding fusion protein (self-iterating factor, or SIF) with an affinity for factors involved in intra lumenal vesicle translocation is thus able to facilitate its own sorting into an exosome for intercellular movement when the SIF is bound to the construct. As the construct codes for more copies of the SIF and can replicate by borrowing DNA replication enzymes from its host cell, this process of exosomal movement of the genetic construct is self-perpetuating. Likewise, with the use of other protein-encoding genes on the genetic construct, the self-iterating spread of a protein by means of exosomes can also be facilitated. 
     
    
     
       DESCRIPTION OF FIGURES 
         [0009]      FIG. 01  demonstrates both phases of process  2 , in which Calcium Ionophore (A23187); hereafter referred to as A23187 in the description and figures, is added to a cell in order to induce exosome release. In the second phase, an exosome containing RNA and protein but not DNA is shown. Process  1 , not shown, may refer to a standard method of preparing an untransfected cell culture to begin an experiment with. 
           [0010]      FIG. 02  illustrates both phases of process  10 , in which A23187 causes a recently released endosome to fold inward, creating an ILV. Process  11  also takes place at the site of ILV formation. 
           [0011]      FIG. 03  also shows process  11 , in which A23187 works with a SIF to cause a genetic construct to enter an ILV. It also shows processes  15  and  17 , where type 1 and type 2 exosomes are released at the plasma membrane, respectively. 
           [0012]      FIG. 04  depicts the three phases of process  4 , in which a liposome containing a SIF-construct complex fuses with an exosome to create an exosome loaded with a SIF-construct complex. 
           [0013]      FIG. 05  demonstrates processes  6 - 9 , in which exosomes loaded with SIF-construct complexes reach target cells and deliver their SIF-construct complexes. They may reach their target cells by being introduced to a cell culture (process  6 ) if the self-iterating cycle is being started. Alternatively, the exosomes may reach their target cells by movement through the extracellular space (process  7 ) if the self-iterating cycle is taking place. In processes  8  and  9 , cells which have received SIF-construct complexes experience replication of the SIFs and constructs. 
           [0014]      FIG. 06  illustrates processes  19 - 22 , in which exosomes are brought into contact with liposomes containing the genetic construct. After fusion of the membranes, the result is exosomes containing the genetic construct. These exosomes can then be added to the target cells such that fusion of the membranes results in cells containing the genetic construct. 
           [0015]      FIG. 07  shows the 22 processes involved with the patent and depicts their relation to the previously mentioned  FIGS. 01-06 . Each process relates to a specific figure, with each figure showing multiple processes. Specifically, the self-iterating cycle is  FIGS. 02-05  repeating themselves.  FIG. 07  references  FIGS. 01-06 , as it places all of them in a single context. 
       
    
    
     DETAILED DESCRIPTION 
       [0016]      FIG. 01  represents processes  0701  through  0703 , as shown in  FIG. 07 . In the following description, process numbers in the individual figures may appear as numbers from  01 - 22 . These numbers may also be referred to with a prefix of “07” preceding the number of the process since in  FIG. 7 , the reference numbers are presented in this manner. Process  0701  is to prepare an untransfected cell culture. A single cell [ 0110 ] is shown in both phase  1  and  2  of process  0702 . In phase  1 , the various components of this typical eukaryotic cell [ 0110 ] are labeled. The plasma membrane [ 0111 ] surrounds the cytosol [ 0107 ], which contains the golgi apparatus [ 0112 ], a mitochondrion [ 0115 ], and the nucleus [ 0120 ]. Outside the cell is a sample molecule of calcium ionophore (A23187) [ 0105 ]. 
         [0017]    The golgi apparatus [ 0112 ] consists of phospholipid bilayer membranes [ 0112 ] enclosing several membrane-bound vesicles [ 0113 ]. Within the internal spaces of the golgi apparatus, a variety of proteins exist [ 0114 ]. 
         [0018]    The mitochondrion consists of two closely packed phospholipid bilayer membranes [ 0115 ] enclosing a small intermembrane space and the interior mitochondrial matrix [0119]. The interior membrane is shaped into folds called cristae [ 0116 ] that point inward within the mitochondrial matrix [ 0119 ]. The mitochondrial matrix contains a mtDNA (mitochondrial DNA) plasmid [ 0118 ], ribosomes [ 0117 ], and proteins (not shown). 
         [0019]    The nucleus [ 0120 ] consists of the nuclear envelope [ 0121 ], the nuclear pores [ 0122 ], DNA molecules [ 0123 ], among other entities. Also included within the nucleus are the RNA molecules [ 0124 ] and protein components [ 0125 ] of the nucleolus, as well as enzymes such as RNA polymerase [ 0126 ]. Outside of the nucleus, the rough endoplasmic reticulum [ 0127 ] has an internal lumen [ 0128 ] and ribosomes on its exterior [ 0129 ]. 
         [0020]    Molecules of A23187, such as the one shown [ 0105 ], enter the cell during process  0702  [ 0106 ]. According to research by Valadi et al., placing HMC-1 cells in a 2 uM solution of A23187 for 30 minutes stimulates exosome release. Thus, A23187 is used as the sample means by which exosome release takes place. 
         [0021]    A sample exosome [ 0153 ] is shown in phase  2  of process  0702 , although this also represents the beginning of process  0703 , in which exosomes containing no DNA (unloaded exosomes) are generated. The interior of the exosome [ 0145 ] is surrounded by a phospholipid bilayer [ 0150 ] with an inner [ 0152 ] and outer [ 0151 ] component. Within the exosome exist molecules of RNA [ 0140 ] and protein [ 0155 ]. 
         [0022]      FIG. 02  shows how intra lumenal vesicles (ILVs), the predecessors of type lexosomes, begin to form. Type lexosomes correspond to the ILVs of multivesicular bodies (MVBs) upon fusion of the outer membrane of a MVB with the plasma membrane of a cell, whereas type 2 exosomes form from endosomal patches of the plasma membrane budding outwards. As explained in  FIG. 07 ,  FIG. 02  corresponds to processes  0710  and  0711 . In phase lof process  0710 , the rough endoplasmic reticulum [ 0227 ] begins to bud off an endosome [ 0231 ] containing protein [ 0230 ]. As previously shown, the rough endoplasmic reticulum has an internal membrane-bound lumen [ 0228 ] and has ribosomes on its exterior [ 0229 ]. In the nearby cytosol, RNA molecules are present [ 0223 ,  0224 ]. 
         [0023]    In phase  2  of process  0710 , A23187 [ 0206 ] facilitates the infolding [ 0245 ] of the endosomal membrane [ 0235 ] in order to initiate the creation of an ILV. The example protein [ 0235 ] within the endosomallumen [ 0236 ] represents the many proteins that may exist within the endosomallumen [ 0236 ]. The endosomal membrane [ 0235 ] is a phospholipid bilayer with an inner [ 0238 ] and outer [ 0237 ] component. RNA molecules [ 0240 ,  0241 ] from the cytosol enter the ILV [ 0246 ], as shown. When the ILV becomes an exosome, these RNA molecules [ 0240 , 0241 ] are referred to as exosomal shuttle RNA (esRNA). 
         [0024]      FIG. 03  corresponds to processes  11 - 18 . In particular, process  0711  is related to both  FIG. 02  and  FIG. 03 , as both illustrate cellular cargo [ 0240 / 0241 ,  0355 / 0356 / 0357 ] entering an ILV [ 0246 , 0345 ,  0346 ]. In  FIG. 03 , A23187 [ 0306 ] facilitates the infolding [ 0358 ] of the external membrane of the MVB [ 0335 ], which is a phosopholipid bilayer [ 0337 ,  0338 ]. Protein from the cytosol [ 0355 ] may then enter [ 0345 ] the newly forming ILV [ 0358 ]. In the case of the genetic construct—pGFP in this example—[ 0356 ], the SIF [ 0357 ] facilitates the entry [ 0346 ] of the SIF-construct complex [ 0356 ,  0357 ] into the newly forming ILV 
         [0025]    The MVB may already contain protein [ 0330 ] inside of its intermembrane space [ 0336 ], as shown. The phospholipid bilayer [ 0351 ,  0352 ] membrane [ 0350 ] of the already-formed ILV [ 0345 ] surrounds the RNA molecules [ 0323 ,  0324 ] that will eventually become esRNAs [ 0340 ,  0341 ]. The above information relates to phase lof process  0711 . 
         [0026]    In process  12 , the MVB [ 0335 ] moves through the cytosol to reach the plasma membrane [ 0311 ], where it fuses (process  0713 ) and releases its contents [ 0368 ,  0369 ]—two ILVs that become exosomes in processes  14  and  15 . Alternatively, endosomal patches of the cellular membrane [ 0311 ] may spontaneously release type 2 exosomes [ 0370 ] in process  0717 . In this way, the second diagram of  FIG. 03  represents processes  15  and  17 . In processes  16  and  18 , the two types of exosomes [ 0368 , 0369 ,  0370 ] move outward into the extracellular space [ 0364 ]. Both types of exosomes [ 0368 ,  0369 , 0370 ] have a phospholipid bilayer [ 0361 ,  0362 ] membrane [ 0363 ] enclosing an exosomallumen [ 0360 ]. Exosomes [ 0368 ,  0369 ,  0370 ] may contain both esRNA [ 0340 ,  0341 ,  0371 ] and protein [ 0365 ,  0372 ]. As shown, the content of any single exosome [ 0368 ,  0369 ,  0370 ] may vary significantly. 
         [0027]    An important feature of  FIG. 03  is that in process  0711 , the SIF [ 0357 ,  0367 ] facilitates the entry of a cytosolic genetic construct [ 0356 ] into a newly forming ILV [ 0358 ]. Through processes  12 - 16 , the ILV containing the SIF-construct complex [ 0356 / 0357 ,  0366 / 0367 ] eventually becomes an exosome [ 0368 ] loaded with the SIF-construct complex [ 0366 ,  0367 ].  FIGS. 04 and 05  depict how an exosome may eventually deliver its SIF-construct complex [ 0366 ,  0367 ] to another cell, where it may replicate in process  0709 , thus creating a self-iterating cycle ( FIGS. 02-05 ) of DNA transfer. This ultimately has the potential to become a vector which will promote the spread of a genetic construct throughout the entirety of a multicellular organism in a non-viral manner. 
         [0028]      FIG. 04  illustrates the three phases of process  0704 , which result in process  0705 , the generation of exosomes [ 0454 ] loaded with SIF-construct complexes [ 0456 ,  0457 ]. Using a lipofection reagent, liposomes [ 0475 ] which contain SIF-construct complexes [ 0456 ,  0457 ] in their lumen [ 0476 ] are generated. These liposomes [ 0475 ] have membranes [ 0477 ] which are phospholipid bilayers with inner [ 0479 ] and outer [ 0478 ] components. As shown in  FIG. 01 , the addition of A23187 to cells can result in the creation of exosomes [ 0153 ,  0453 ] which are not loaded with DNA. The addition of these lipsomes constitutes phase lof process  0704 . However, lipofection of exosomes [ 0480 ] in phase  2  of process  0704  can remedy this situation. As this occurs [ 0480 ], the SIF-construct complexes [ 0456 , 0457 ] enter the exosomes [ 0453 ]. During phase  2 , the outer layers [ 0478 ,  0451 ] and the inner layers [ 0479 ,  0452 ] of the liposome and exosome fuse such that the two phospholipid bilayers [ 0477 ,  0450 ] become one. In this way, the cargo of the liposome [ 0456 ,  0457 ] and exosome [ 0440 ,  0445 ] become united within one biological vesicle [ 0480 ] during phase  2 . In phase  3  of process  0704 , the fusion is complete and the result [ 0454 ] culminates in process  0705 , which is the generation of composite exosomes [ 0454 ] containing SIF-construct complexes [ 0456 ,  0457 ]. 
         [0029]      FIG. 05  corresponds to processes  6 - 9 . Exosomes [ 0554 ,  0569 ] generated in process  0705  are shown to the left of a cell [ 0510 ]. In order to start the self-iterating cycle ( FIGS. 02-05 ), exosomes [ 0454 ] containing SIF-construct complexes [ 0554 ] may be added to cells [ 0581 ] in process  0706 . If the cycle ( FIGS. 02-05 ) is already in progress, then exosomes [ 0569 ] from other cells [ 0311 ] may reach [ 0582 ] target cells [ 0510 ] in process  0707  by means of exosome movement. As previously shown, exosomes can contain both RNA [ 0540 ] and protein [ 0555 ] content. Processes  6  and  7  culminate in the fusion [ 0581 ,  0582 ] of exosomal membranes [ 0556 ,  0557 ] with the plasma membranes [ 0511 ] of target cells [ 0510 ]. 
         [0030]    Process  0708  in  FIG. 05  refers to the generation of cells transfected [ 0590 ] with SIF-construct complexes [ 0566 ,  0567 ], the restart step in the self-iterating cycle ( FIGS. 02-05 ). This, as previously stated, occurs as exosomes [ 0554 ,  0569 ] deliver their SIF-construct complexes [ 0566 ,  0567 ] to the target cells [ 0510 ,  0590 ] through exosome movement [ 0581 ,  0582 ] and membrane fusion [ 0556 ,  0557 ,  0511 ]. This also represents the first phase of process  0709 , in which SIF-construct complexes in cells replicate. Alternatively, process  0708  may be reached from process  0701   if  the cells are lipofected with SIF-construct complexes. 
         [0031]    Phase  2  of process  0709  in  FIG. 05  refers to the replication of the SIF-construct complexes [ 0566 ,  0567 ]. As previously mentioned, the genetic construct [ 0566 ] codes for the SIF [ 0567 ], among other gene products. Over time, the presence of the genetic construct [ 0566 ] in the cytosol will lead to the transcription of the SIF mRNA and the translation [ 0568 ] of this mRNA into the SIF protein [ 0567 ] at a ribosome [ 0529 ]. Furthermore, the SIF [ 0567 ], like most DNA-binding proteins, has a variable affinity for its binding site and can dissociate at times. When the genetic construct [ 0566 ] is not bound to its SIF [ 0567 ], it is unlikely to be taken into an ILV and transported to another cell via exosomal shuttling. This allows for the DNA replication enzymes of the host cell [ 0510 ,  0590 ,  0595 ] to produce a new copy of the genetic construct [ 0586 ]. In this way, new copies of the SIF [ 0587 ] and genetic construct [ 0586 ] can be produced. In order to prevent this process from continuing indefinitely, the SIF-encoding gene on the genetic construct [ 0566 ,  0586 ] contains signals for methylation that will reduce the expression [ 0568 ] of that particular gene over time. Ideally, SIF production [ 0568 ] will halt when nearly every cell [ 0590 ,  0595 ] in the culture or multicellular organism has at least one copy of the genetic construct [ 0566 ,  0586 ] such that expression of the genetic construct [ 0566 ,  0586 ,  0529 ] will be nearly universal. This part of  FIG. 05 , process  0709 , completes the self-iterating cycle, which is defined as  FIGS. 02-05  repeating until expression of the genetic construct&#39;s SIF-encoding gene [ 0566 ,  0568 ,  0567 ] declines significantly. 
         [0032]      FIG. 06  represents the one-time variant of the exosomal vector [ 0654 ]. Specifically, the sequence of processes  19  through  22  results in the transfecting [ 0683 ] of target cells [ 0685 ] with copies of the genetic construct [ 0656 ]. In phase lof process  0719 , the lipofection of exosomes [ 0653 ] by means of liposomes [ 0675 ] carrying the genetic construct [ 0656 ] is shown. As previously explained, both liposomes [ 0675 ] and exosomes [ 0653 ] have membranes [ 0677 ,  0650 ] which are phospholipid bilayers with inner [ 0679 ,  0652 ] and outer [ 0678 ,  0651 ] components. Also, exosomes typically contain both RNA [ 0640 ] and protein [ 0655 ] content. In process  0719 , the fusion [ 0681 ,  0691 ] of the membranes [ 0677 ,  0650 ] of the liposomes [ 0675 ] and exosomes [ 0653 ] combines the two interiors [ 0682 ,  0645 ] into one new lumen [ 0686 ]. This brings the overall procedure to phase lof process  0720 , the generation of newly created exosomes [ 0680 ] loaded with the genetic construct [ 0686 ] in their interior [ 0697 ]. In processes  21  and  22 , these exosomes [ 0654 ] loaded with the genetic construct [ 0686 ] are added [ 0683 ] to the target cells [ 0685 ] such that the two plasma membranes [ 0691 ,  0611 ] rearrange themselves [ 0684 ] and fuse together into a single membrane [ 0612 ]. As this occurs, the exosomallumen [ 0697 ] becomes one with the cytosol [ 0687 ,  0688 ]. Furthermore, this action delivers the genetic construct [ 0686 ] in the lumen [ 0697 ] of the exosome [ 0654 ] into the cytosol [ 0687 ,  0688 ] such that it [ 0686 ,  0696 ] now resides in the cytosol [ 0688 ] of the composite cell [ 0690 ]. 
         [0033]      FIG. 07  is the flowchart that describes all 22 of the above processes in a single page. For a one-time transfection, one can simply follow  FIGS. 01 and 06  [ 0153 ; 0653 ; 0680 / 0654 ,  0681 ,  0683 , 0696 ]. The sample chemical used for exosome release, A23187 [ 0105 / 0106 ,  0206 ], is used in both  FIG. 01  and  FIG. 02 . In  FIGS. 02 and 03 , the first steps [ 0231 ,  0206 ,  0246 ] leading to exosome release [ 0368 ,  0369 ,  0370 ] occur as part of a self-iterating cycle ( FIGS. 02-05 ). process  0711  [ 0346 ], in which SIF-construct complexes [ 0356 ,  0357 ] enter the ILVs [ 0358 ] of MVBs [ 0353 ], is related to both  FIGS. 02 and 03 , as both feature the activity [ 0245 ,  0358 ] of A23187 [ 0206 ,  0306 ]. In  FIG. 03 , both types of exosomes [ 0368 / 0369 ,  0370 ] are shown outside [ 0364 ] of a cell [ 0311 ] just as process  0705  [ 0368 ,  0369 ,  0370 ] is reached from processes  15 / 16  [ 0399 ] and  17 / 18  [ 0398 ]. 
         [0034]    In  FIG. 04 , process  0704  [ 0481 ], the alternative method of reaching process  0705  [ 0480 ,  0454 ], is shown. This serves as the connection between processes  3  [ 0153 ] and  5  [ 0454 ], where  FIG. 01  links into the self-iterating cycle of  FIGS. 02-05 . Finally,  FIG. 05  completes the self-iterating cycle of  FIGS. 02-05  through processes  6 - 9  [ 0581 ,  0582 ,  0590 ,  0568 / 0595 ], in which exosomes [ 0554 ,  0569 ] deliver their SIF-construct complexes [ 0566 ,  0567 ] to target cells [ 0510 ], where they can then replicate [ 0568 ] before being delivered [ 0346 ,  0366 / 0367 ,  0369 ,  0582 ] to other cells [ 0510 ] as the cycle ( FIGS. 02-05 ) starts again. In this way, DNA, RNA, and protein transfer to an entire cell culture, tissue, organ, organ system, or even organism may take place for experimental, research, therapeutic, or diagnostic purposes.