WO2017070392A1 - Apparatus and methods for production of acellular tissues for organ regeneration - Google Patents

Abstract

Disclosed are methods for decellularizing an isolated organ, or a portion thereof, by mechanically agitating the isolated organ with a detergent-containing fluid that removes the cellular membrane surrounding the isolated organ, in the presence of a regulatable source of air to control the removal of detergent from the decellularized organ, while maintaining the extracellular matrix and scaffold. Also disclosed are methods for producing mesoparticle-infused acellular tissues for generation of mammalian organs and tissues suitable, inter alia, for human transplantation

Description

DESCRIPTION

APPARATUS AND METHODS FOR PRODUCTION OF ACELLULAR TISSUES FOR ORGAN

REGENERATION

BACKGROUND OF THE INVENTION

CROSS -REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Application No. 62/244,137, filed October 20, 2015 (pending; Atty. Dkt. No. 37182.200PV01); the contents of which is specifically incorporated herein in its entirety by express reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

[0003] Not Applicable.

FIELD OF THE INVENTION

The present invention generally relates to the field of medicine, and in particular, to organ regeneration and transplantation. Disclosed are apparatus and methods for improving decellularization of organ tissues while maintaining the extracellular matrix and scaffold. In exemplary embodiments, a novel method is provided for the decellularization of whole organs, which improves the preservation of the acellular scaffold. Using nanoparticle-based delivery strategies, the controlled release of one or more active agents may be accomplished to promote proper cellular differentiation, proliferation, and/or growth for the purpose of regenerating a functional organ.

DESCRIPTION OF RELATED ART

ACELLULAR TISSUE PRODUCTION

[0004] Conventional methods for the decellularization of whole organs include the use of harsh detergents that not only effectively remove the cells from the scaffold, but also damage the acellular scaffold as well.

[0005] Established methods for the decellularization of organs include the use of harsh detergents, typically for a duration of 7-10 days. This prolonged exposure to detergents effectively removes not only cells, but also critical scaffold materials such as fibrin, elastin and collagen.

[0006] Conventional methods for the decellularization of whole organs include the use of harsh detergents that not only effectively removes the cells from the scaffold, but also damages the acellular scaffold as well. Existing methods also teach that the 5 acellular lungs should be washed with DI water and/or dilute alcohol solutions to remove excess detergent. This is performed by perfusing with a syringe or a pump down the pulmonary artery, followed by external rinsing. However, these methods have proven to be timely and largely ineffective. l o DEFICIENCIES IN THE PRIOR ART

[0007] Organ transplantation represents an important way of restoring function when an organ is irreparably damaged; the surgical process has saved tens of thousands of lives worldwide. However, problems exist when there is a transfer of biological tissue form one individual to another. Organ rejection is a significant risk associated with

15 transplantation, even when there is indication of a good histocompatability match.

Immunosuppressive drugs such as cyclosporin and FK506 are usually given to the patient to prevent rejection. These immunosuppressive drugs however, have a narrow therapeutic window between adequate immunosuppression and toxicity. Prolonged immunosuppression can weaken the immune system, which can lead to a threat of

20 infection. In some instances, even immunosuppression is not enough to prevent organ rejection.

[0008] Another major problem of transplantation, is the availability of donor organs. In the United States alone there are about 50,000 people on transplant waiting lists, many of whom will die before an organ becomes available.

25 [0009] Existing literature supports the need for a new decellularization process that reduces the degradation of the acellular scaffold since it has been demonstrated that the tissue matrix plays a critical role in the appropriate differentiation and proliferation of native cell types needed for a functional organ.

30 BRIEF SUMMARY OF THE INVENTION

[0010] The present invention overcomes these and other inherent limitations in the art by providing, in a general sense, methods for the decellularization of a whole organ, which improves the preservation of the acellular scaffold. The present disclosure also provides compositions and methods for integrating nano-based delivery strategies to provide the controlled release of active agents to promote proper cellular differentiation, proliferation, and growth for the purpose of regenerating a functional organ.

[0011] In certain aspects, the present disclosure provides methods that reduce exposure of the tissues to detergents for preferably less than 3 or 4 hours. Furthermore, methods for controlling the delivery of one or more active agents (for example, and without limitation, such as growth factors, antibiotics, etc.) to the acellular scaffold have been employed post-decellularization to improve the acellular construct to better support organ regeneration.

[0012] To further advance the regenerative potential of the acellular scaffold, in related embodiments, pluralities of nanovector delivery particles may also infused within the scaffold matrix, which can be pre-selected to deliver one or more of the active agents in timed-release and/or in sequential manner to promote proper cell growth and function of the resulting tissue scaffold and development of the functional organ.

[0013] One of the biggest challenges presently in the art following decellularization has been the difficulty in halting continued damage to the acellular scaffold by the presence of residual detergent. Previously, removal of the residual detergent was accomplished by multiple repeated washing of the tissue by perfusion with large volumes of water or other buffer solutions relative to the sign of the tissue. Not only was the process tedious, cumbersome, and time consuming, it has not been effective for the preservation of certain tissues (such as mammalian lung in particular) because damage caused by residual detergent often rendered the scaffold unusable.

[0014] In an important aspect, the present invention overcomes this prior-art limitation by providing an apparatus and method for producing a superior decellularized ECM scaffold organ tissue. By integrating an aeration mechanism in a conventional bioreactor apparatus, the surface tension of the detergent bubbles can be exploited to quickly remove the detergent from the bioreactor AND the acellular lung tissues. This bubbling action not only mechanically agitates the tissue to further facilitate "washing" of detergent from the acellular tissue, but it also effectively removes the detergent from the perfusate solution itself. In a particular embodiment, a bioreactor apparatus is provided that integrates a "bubbling bar" (i.e., an aeration tube, stone, fritted or scintered glass element, etc.) operably connected to a suitable air source, which effectively and simultaneously removes detergent from both the acellularized organ, and from the bioreactor itself through the creation of a bubbling or foaming effect that quickly diffuses excess detergent from the surface of the tissue or organ being prepared for acellular scaffolding.

[0015] In general, the invention pertains to methods of producing decellularized organs, using an isolated organ or a part of an organ and a series of extractions that removes the cell membrane surrounding the organ, or part of an organ, and the cytoplasmic and nuclear components of the isolated organ, or part of an organ.

[0016] Accordingly, in one aspect, the invention provides a method for producing a decellularized organ comprising:

[0017] mechanically agitating an isolated organ to disrupt cell membranes without destroying the interstitial structure of the organ;

[0018] treating the isolated organ in a solubilizing (i.e., detergent-containing) fluid at a concentration effective to extract cellular material from the organ without dissolving the interstitial structure of the organ; and

[0019] washing the isolated organ in a washing fluid to remove cellular debris without removing the interstitial structure of the organ until the isolated organ is substantially free of cellular material, to thereby produce a decellularized organ.

[0020] The method preferably also includes providing an aeration source in the organ decellurization chamber (as seen schematically in FIG. 1 and FIG. 12) to facilitate the controlled removal of the detergent-containing washing fluid from the decellularized organ in an expedited fashion to minimize untoward degradation of the resulting acellular scaffold. Preferably the aeration source is a "bubbler" or aeration stone that facilitates agitation of the detergent fluid to form miscelles, which are then removed from the treatment chamber, thereby reducing the concentration of residual detergent in the washing medium.

[0021] Optionally, the method can further comprise drying the decellularized organ, which can the optionally be stored at a suitable temperature, or equilibrated in a physiological buffer prior to use.

[0022] Optionally, mechanical agitation of the solution comprising the isolated may be employed, which further comprises placing the isolated organ in a stirring vessel having one or more stirring devices (i.e. paddles) which can rotate at a speed ranging from about 25 to 250 revolutions per minute (rpm).

[0023] In one embodiment, the step of mechanically agitating the isolated organ occurs in a fluid selected from the group consisting of distilled water, physiological buffer and culture medium.

[0024] In one embodiment, the step of treating the isolated organ in the solubilizing fluid also occurs in a stirring vessel.

[0025] In a preferred embodiment, the solubilizing fluid is an alkaline solution having a detergent. In more preferred embodiment, the alkaline solution is selected from the group consisting of sulphates, acetates, carbonates, bicarbonates and hydroxides, and a detergent is selected from the group consisting of Triton X-100, Triton N-101, Triton X- 114, Triton X-405, Triton X-705, and Triton DF-16, monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 80), polyoxyethylene-23-lauryl ether (Brij 35), polyoxyethylene ether W-l (Polyox), sodium cholate, deoxycholates, CHAPS, saponin, n-Decyl βΡ-D-glucopuranoside, n-heptyl β-D glucopyranoside, n-Octyl a-D- glucopyranoside and Nonidet P-40. In the most preferred embodiment, the solubilizing solution is an ammonium hydroxide solution having Triton X-100.

[0026] In one embodiment, the step of washing the isolated organ also occurs in an aeration vessel. The washing fluid can be selected from the group consisting of distilled water, physiological buffer and culture medium.

[0027] In another aspect, the invention features a method for producing a decellularized organ comprising:

[0028] mechanically agitating an isolated organ in distilled water to disrupt cell membranes without destroying the interstitial structure of the organ;

[0029] treating the isolated organ in an alkaline solution having a detergent at a concentration effective to extract cellular material without dissolving the interstitial structure of the organ;

[0030] washing the isolated organ in distilled water to remove cellular debris without removing the interstitial structure of the organ until the organ is substantially free of the cellular material, to thereby produce a decellularized organ.

[0031] In a preferred embodiment, the decellularized organ is a mammalian lung, and preferably, a human lung.

BRIEF DESCRIPTION OF THE DRAWINGS:

[0032] The following drawings form part of the present specification and are included to demonstrate certain aspects of the present disclosure. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0033] For promoting an understanding of the principles of the invention, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will, nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the invention relates. The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

[0034] FIG. 1 shows a schematic of a bioreactor used to decellularized pig lung, notice that the parts of the organ, such as the pulmonary artery (PA) and the trachea, as well as the lung itself, are represented by simple shapes;

[0035] FIG. 2 and FIG. 3 show an exemplary bioreactor with tubes disposed as described in the prior schematic (FIG. 1); FIG. 2 shows the bioreactor is connected to a porcine lung; FIG. 3 is the empty bioreactor chamber itself;

[0036] FIG. 4A and FIG. 4B show screenshots of a Hamilton-C2 ventilator while operating in constant volume mode; APV_cmv (FIG. 4A) or in constant pressure mode; PCV+ (FIG. 4B);

[0037] FIG. 5 shows exemplary embodiments in accordance with one aspect of the present invention involving lungs #4 and #5 after the decellularization process. Notice the numerous delaminations present on the surface. The presence of these delaminations did not constitute a significant issue, since it was noticed that few of those were present also just after lung grafts, indicating that they may be a minor concern also for healthy organs. Although delamination could be tolerated, lung 4 showed too many of them, thus leading to a further improvement of the final protocol; Lungs 5, 6 and 7 were treated with different protocols, but based on the same one employed for lung 4. Different sequence of 0.002 % Dextrose, 0.2 % SDS and 2 % SDS solution were tried; lung #5 was treated for almost one entire week with Dextrose but the employment of SDS ended up being necessary since Dextrose alone was insufficient to remove cells;

[0038] FIG. 6 shows an exemplary embodiment in accordance with one aspect of the present invention illustrating lung #5 after the decellularization. It is evident, in this case, how SDS damaged the ECM too much; it appeared almost transparent in certain areas;

[0039] FIG. 7 shows an exemplary embodiment in accordance with one aspect of the present invention illustrating lung #6 after the decellularization. This situation instead it really differs from the previous one; here the lung was still pink after the treatment, meaning that the SDS treatment was inefficient;

[0040] FIG. 8 shows an exemplary embodiment in accordance with one aspect of the present invention illustrating lung #7 after the decellularization. In this study, the lung appeared incompletely decellularized;

[0041] FIG. 9 is a photograph of lung #8 after the decellularization. It has a homogeneous color and it showed all the structures typical of the organ when dissected;

[0042] FIG. 10 is a photograph of lung #11 before the decellularization while it was still attached to the ventilator;

[0043] FIG. 11 shows a photograph of lung #11 just after the ventilation step that follows the end of the decellularization process;

[0044] FIG. 12 shows a schematic representation of the bubbling technique starting from the scheme shown above. Bubbles coming from the bottom of the bioreactor, passing through the red tube system, should cause SDS to self-assemble, and go on the surface of the rinsing solution; there, thanks to a built-in pressure gradient, they leave the reactor throughout the yellow tube system;

[0045] FIG. 13 shows a plot of average SDS concentration of the five lungs at different moments of the bubbling rinse;

[0046] FIG. 14A, FIG. 14B, and FIG. 14C show the results of various analyses of the lungs shown above. In FIG. 14A, a plot of average SDS concentration of the five lungs at different moments of the bubbling rinse is compared to the concentration used during the decellularization. In FIG. 14B, average SDS concentration of samples from rinsing solution is shown. Notice how the SDS concentration of drained bubbles and overflowed solution (2 columns on the right) are the highest, while the last sample collected (i.e., 27 hrs) is the lowest. FIG. 14C is a plot that shows the ratio between the SDS (mg) found in samples and the mass (mg) of the samples for all the five lungs treated with the good protocol. Each column shows the average value and the standard error of the samples coming from the five different lungs but from the same lobes;

[0047] FIG. 15 shows the ratios between SDS (mg) present in the sample and the mass (mg) of the samples it is pretty straightforward that lungs washed with bubbling technique (i.e. lungs 8, 9, 10, and 11) (see also FIG. 10 and FIG. 11) have really low ratio values compared with the ones of lungs rinsed with different techniques (i.e., lungs 4 and 7 were the best ones among the others). The legend on the right simply indicates from which zone lobe of the lung samples were collected;

[0048] FIG. 16 the curved shows the amount of protein during the decellularization process when 0.002% was used; it shows the average result evaluated for the 5 lungs treated with the final protocol. Notice the plateau that tends to from after a couple of hours since the beginning of the process or change of 0.002% Dextrose solution;

[0049] FIG. 17A and FIG. 17B show the average ratio between the protein amount and the weight of the relative tissue sample and the standard error; and the average ratio between the protein amount and the weight of the relative tissue sample including the control lungs, respectively;

[0050] FIG. 18A and FIG. 18B show physical loading of the growth factor inside the cavities of the mesoporous silicon particle, yellow arrows (FIG. 18A); and the chemical attachment of growth factor throughout functionalization of the silicon particles; blue arrows (FIG. 18B);

[0051] FIG. 19A, FIG. 19B, and FIG. 19C show IVIS images of a control porcine lung not perfused with particles (FIG. 19A); a normal porcine lung perfused with rhodamine-loaded MSVs (FIG. 19B); and a decellularized porcine lung not perfused with particles (FIG. 19C);

[0052] FIG. 20 shows the number of particles per mg of tissue found using the ICP technique: starting from the left, it reports the value for the experiments done, respectively with discoidal perfused in Hespan®, discoidal in Hespan® and blood solution, spherical in Hespan® solution and spherical in Hespan® and blood solution. It was straightforward to see how the discoidal particles in both cases exceeded the values of the spherical ones;

[0053] FIG. 21A, FIG. 21B, and FIG. 21C show SEM images of an illustrative sample. In FIG. 21A the area delimited in green is the part of the sample where the tissue was found to be torn; FIG. 21B shows a higher magnification SEM image of the area delimited in green in FIG. 21A, while FIG. 21C shows a still higher magnification SEM image of the area delimited in green in FIG. 21 A; and

[0054] FIG. 22 shows the curve obtained while loading different masses (pg) of fluorescent albumin in 5 million MSVs having a diameter of 2.6 pm.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0055] Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0056] End-stage lung disease is still today a cause of death and the only way to survive is throughout lung transplantation. Although lung transplantation is a surgical operation that became quite common it still presents several related issues: the significant shortage of lungs for transplantation is the main one, this is then related with the need for immunosuppression and thus with transplanted lung rejection by the host organism. In the United States, end-stage lung disease is the fourth cause of death (Xu et al, 2007). Even after transplantation the chance for surviving and keeping the graft function are not so high: in fact one third of the patients who received a lung transplant undergo a severe rejection episode, and only the 50% of the patients that survives the first here will be able to preserve graft function after 5 years (Christie et al, 2010). This data are significant if one wants to underline the importance of finding a remedy to this issue: the main tendency, recently, has been the design and development of artificial scaffold which give the possibility to grow cells on to them. Being able to use a lung scaffold well-formed so that it allows for stem cell growth, differentiation and, finally, a complete organ recellularization is the ultimate goal of tissue engineering; it will give the possibility to overcome the most significant hurdles like transplanted organ rejection and donor organ shortage.

[0057] Bioartificial lung creation characterized the first attempt to the goal, and it is still on going in many research laboratories, but, at the moment, the major efforts are focused on the creation of scaffolds starting from dead-animal organs throughout a decellularization process, i.e., a procedure aimed to remove all cells from an organ, or a piece of that, and leave the ECM (Song and Ott, 2012). Literature shows several efforts made in accomplishing this goal and the importance of having a good scaffolds is of primary importance especially when dealing with recellularization; as Joaquin Cortiella and his group has proved on decellularized lung scaffolds (Nichols et al, 2012), preserving all the typical anatomical, chemical and morphological features of a real lung helps the differentiation and the three dimensional proliferation of stem cells seeded on the scaffold more than artificial scaffolds (Song and Ott, 2012; Nichols et al, 2009). The matrix chemical composition and morphology play a crucial role in guiding seeded stem cells in their differentiation and proliferation and since lung is a complex organ, arranged by different type of cells (such as type I AEC and type II AEC and others), and have a complex, branched morphology it is difficult to reproduce these characteristic in an artificial one, for not even mentioning the bio-compatibility of those.

[0058] Although these wonderful aspects of decellularized lungs, it must be said that they are not exempt form issues, indeed an harsh decellularization may damage the organ too much and bring to different or, even worse, not wanted results that lead to an incomplete recellularization; thus the present work improves this process by delivering growth factors to seeded stem cells (Song and Ott, 2012).

[0059] Tissue engineering and transplant surgery are increasingly focused on decellularized matrix scaffold. End-organ failure is an healthcare challenge still unresolved; indeed the only way to overcome this kind of diseases is a successful organ transplant, but the shortage of organs suitable for a transplant and the rejection response started by the host body are crucial factors of transplant failure. One way to deal with these issues has been recognized in the decellularization and subsequent recellularization of natural organs donated by deceased patients or compatible animals. Decellularization means the process by which, throughout the employment of specific solvents and solutions, the removal of all cells present in the organ and major histocompatibility complex (MHC) antigens is carried out. In 2008, the successful transplantation of human trachea, previously decellularized and later seeded with epithelial cells and mesenchymal stem-cell-derived chondrocytes was reported (Macchiarini et al, 2008), although the operation regarded a simple part of the more complex whole organ it serves as a first confirmation that the path followed may be the right one. The general and final purpose of these studies is the realization of a complete and functional organ to place in a patient with end-organ failure disease and since the availability of human donors is not comparable with the amount of requests it is believed that organs derived from compatible animals may be the ultimate goal.

[0060] Although organ decellularization is a relatively new object of study and development, there are already many reports of different techniques and different materials employed to accomplish that. Badylak et al. (2011) gave a detailed review on the most common decellularization protocol developed up to the moment (Crapo et al, 2011). The preservation of the ECM is of vital importance for obtaining proper reconstruction of the whole organ, so it is really important to obtain a state of decellularization in which all original cells and immunogens are removed but where the protein and other materials, such as glycosaminoglycan (GAG), are preserved properly. Decellularization agents can consist of the most different set of materials and solutions as well as physical approach.

THERAPEUTIC AGENTS

[0061] Exemplary therapeutic agents, which may be administered to a subject in need thereof, by incorporation of the agent(s) within one or more populations of the mesoporous silicon particles described herein, include, without limitation, one or more drugs, small molecules, proteins, lipids, nucleic acids, diagnostic markers, and such like. In the practice of the invention, mesoporous silicon nano or micro-particles may preferably be configured into a shape selected from the group consisting of discoidal, spheroid, non-spheroid, oblate spheroid, and combinations thereof. Preferably, the porous particle is fabricated of a porous or mesoporous silicon material that is discoidal in shape.

[0062] An active agent's ability to reach an intended target at a desired concentration is usually affected by a multiplicity of biological barriers. The biological barrier may be, for example, an epithelial or endothelial barrier, such as the blood-brain barrier, that is based on tight junctions that prevent or limit para-cellular transport of an active agent. Cells of the reticulo-endothelial system may also act as a biological barrier against an active agent. The biological barrier may also be represented by a cell membrane or a nuclear membrane of a target cell.

[0063] In some embodiments, mesoporous silicon particles employed herein for delivery of one or more agents to the tissues or organs being prepared for transplantation are able to overcome at least one biological barrier, including one or more biological barriers selected from the group consisting of a hemo-rheology barrier, a reticuloendothelial barrier, a blood-brain barrier, a tumor-associated osmotic interstitial pressure barrier, an ionic- or a molecular-pump barrier, a cell-membrane barrier, an enzymatic - degradation barrier, a nuclear membrane barrier, and combinations thereof.

[0064] In related embodiments, these mesoporous silicon particles may have at least one targeting moiety on its surface specifically directed against a target cell. In some embodiments, the at least one targeting moiety is selected from the group consisting of ligands, antibodies, antibody fragments, peptides, ap tamers, small molecules, and combinations thereof. For example, ligands can be chemically linked to appropriate reactive groups on the surface of the particle. Protein ligands can be linked to amino- and thiol-reactive groups under conditions effective to form thioether or amide bonds respectively. Methods of attaching antibody or other polymer-binding agents to an inorganic or polymeric support are detailed elsewhere (see, e.g., Taylor, 1991).

[0065] Any active agent, a small molecule drug or a biomolecular drug, may be delivered using the mesoporous silicon particles employed herein for delivery of one or more agents to the tissues or organs being prepared for transplantation. In some embodiments, the at least one active agent is a biologically active compound selected from the group consisting of peptides, proteins, therapeutic agents, diagnostic agents, non-biological materials, and combinations thereof. The therapeutic agent may be any physiologically or pharmacologically active substance that can produce a desired biological effect. The therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, an anti-rejection agent, a cytokine, a cytotoxic agent, a nucleolytic compound, an anti-inflammatory compound, or a pro-drug enzyme, which may be naturally occurring, or produced by synthetic or recombinant methods, or by a combination thereof.

[0066] In some embodiments, the therapeutic agent(s) may be a hydrophobic drug or a hydrophilic drug. Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. A cancer chemotherapy agent may be a preferred therapeutic agent. For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician 's Desk Reference and to the reference by Goodman and Gilman (2001).

[0067] In some embodiments, the therapeutic agent may be selected from the group consisting of genes, nucleic acids, shRNAs, siRNAs, microRNAs, DNA fragments, RNA fragments, plasmids, and combinations thereof. In an illustrative embodiment, the therapeutic agent is a siRNA or a microRNA that silences one or more genes expressed by the cancer cells or the tumor. The therapeutic agent can also be applied to engineer the genome of cancer cells and/or stromal cells in the tumor, such as a CRISPR/Cas9 system.

[0068] The composition of the disclosure may be designed, formulated and processed so as to be suitable for a variety of therapeutic and diagnostic uses and modes of administration. [0069] The composition of the disclosure may be administered to a subject, such as a human, via any suitable administration method in order to treat, prevent, and/or monitor a physiological condition, such as a disease. Embodiments of the composition may be particularly useful for organ transplantation.

COMPOUNDS AND PHARMACEUTICAL FORMULATIONS

[0070] The mesoporous silicon particles employed herein may be employed as a single treatment modality, or alternatively may be combined with one or more additional therapeutic, diagnostic, and/or prophylactic agents, including, without limitation, one or more proteins, peptides, polypeptides (including, without limitation, enzymes, antibodies, antigens, antigen binding fragments etc.); RNA molecules (including, without limitation, siRNAs, microRNAs, iRNAs, mRNAs, tRNAs, or catalytic RNAs, such as ribozymes, and the like), DNA molecules (including, without limitation, oligonucleotides, polynucleotides, genes, coding sequences (CDS), introns, exons, plasmids, cosmids, phagemids, baculovirus, vectors [including, without limitation, viral vectors, virions, viral particles and such like]); peptide nucleic acids, detection agents, imaging agents, contrast agents, detectable gas, radionuclides, or such like, and one or more additional agents, or any combination thereof as part of a multifactorial, or multifocal treatment plan for the affected patient.

[0071] Mesoporous silicon particles may also further optionally include one or more additional active ingredients, including, without limitation, one or more transcription factors, immunomodulating agents, immunostimulating agents, neuroactive agents, antiinflammatory agents, chemotherapeutic agents, hormones, so called "trophic factors," cytokines, chemokines, receptor agonists or antagonists, or such like, or any combination thereof.

[0072] The mesoporous silicon drug delivery formulations may also further optionally include one or more additional components to aid, facilitate, or improve delivery of a pro-drug and/or an active metabolite contained therein, including, without limitation, one or more liposomes, lipid particles, lipid complexes, and may further optionally include one or more binding agents, cell surface active agents, surfactants, lipid complexes, niosomes, ethosomes, transferosomes, phospholipids, sphingolipids, sphingosomes, or any combination thereof, and may optionally be provided within a pharmaceutical formulation that includes one or more additional nanoparticles, microparticles, nanocapsules, microcapsules, nanospheres, microspheres, or any combination thereof. [0073] Preferably, the polycation-functionalized nanoporous silicon carriers of the present disclosure will generally be formulated for systemic and/or localized administration to an animal, or to one or more cells or tissues thereof, and in particular, will be formulated for localized administration to a mammal, prior to, during, or following transplantation of one or more tissues, such as a lung.

[0074] Preferably, drug-delivery formulations of the active compounds disclosed herein will be at least substantially stable at a pH from about 4.2 to about 8.2, and more preferably, will be substantially stable at a pH of from about 5 to about 7.5. Preferably, the active ingredient(s) and targeted drugs will be substantially active at physiological conditions of the animal into which they are being administered.

[0075] The present disclosure also provides for the use of one or more mesoporous silicon carriers in the manufacture of a medicament for therapy and/or for the amelioration of one or more symptoms of a disease, disorder, dysfunction, or condition, and particularly for use in the manufacture of a medicament for treating, one or more diseases, dysfunctions, or disorders involving, or arising from transplantation of one or more tissues or organs in a mammal, and, in a human, in particular.

[0076] The present disclosure also provides for the use of one or more of the disclosed polycation-functionalized nanoporous silicon drug delivery systems in the manufacture of a medicament for the transplantation of a mammalian organ, and in particular, in the transplantation of a human lung. In certain embodiments, the invention also includes diagnostic and/or targeting compounds that may be optionally included in or on the surface of the silicon nanoparticle carriers to facilitate improvements in the treatment or prognosis of the organ transplantation. [0077] Another important aspect of this disclosure concerns methods for using the polycation-functionalized nanoporous silicon carriers to facilitate treatment or the amelioration of one or more symptoms of the disease in a mammal having, suspected of having, or at risk for developing such a condition, and in particular for those mammals undergoing organ or tissue transplant. Such methods generally involve administering to a mammal (and in particular, to a human in need thereof), one or more of the disclosed polycation-functionalized nanoporous silicon carriers formulated to contain one or more therapeutic or diagnostic agents, in an amount and for a time sufficient to diagnosis, monitor, treat (or, alternatively, to ameliorate one or more symptoms of) a condition in a mammal to which the composition has been administered. [0078] In certain embodiments, the therapeutic formulations described herein may be provided to the animal as a single treatment modality, as a single administration, or alternatively provided to the patient in multiple administrations over a period of from several hours to several days, from several days to several weeks, or even over a period of several weeks to several months or longer, as needed following organ transplantation as may be needed. In some aspects, it may be desirable to continue the treatment throughout the lifetime of the patient. In other embodiments, it may be desirable to provide the therapy in combination with one or more existing, or conventional, treatment regimens.

THERAPEUTIC KITS

[0079] Therapeutic kits that include one or more of the disclosed therapeutic drug compositions (and instructions for using the kit) also represent an important aspect of the present disclosure. Such kits may further optionally include one or more diagnostic agents, one or more therapeutic agents, or any combination thereof, either alone or further in combination with one or more additional compounds, pharmaceuticals, or such like.

[0080] The chemotherapeutic kits of the present disclosure may be packaged for commercial distribution, and may further optionally include one or more delivery devices adapted to deliver one or more therapeutic composition(s) to an animal (e.g., syringes, injectables, and the like). Such kits typically include at least one vial, test tube, flask, bottle, syringe or other container, into which the mesoporous silicon carrier-based therapeutic composition(s) may be placed, and preferably suitably aliquotted. Where a second pharmaceutical compound is also provided, the kit may also contain a second distinct container into which this second composition may be placed. Alternatively, a plurality of mesoporous silicon carrier-based compositions as disclosed herein may be prepared in a single mixture, including those prepared as a suspension or in solution, and may be packaged in a single container, such as a vial, flask, syringe, catheter, cannula, bottle, or other suitable containment.

[0081] The kits of the present disclosure may also typically include a retention mechanism adapted to contain or retain the vial(s) or other container(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) or other container(s) may be retained to minimize or prevent breakage, exposure to sunlight, or other undesirable factors, or to permit ready use of the composition(s) included within the kit.

PREPARATION OF MEDICAMENTS

[0082] Another important aspect of the present disclosure concerns methods for using the disclosed mesoporous carriers as agents for delivering therapeutic or diagnostic compounds to selected cells or tissues or organs of a vertebrate mammal, and particularly in a mammal undergoing a tissue or an organ transplant, such as a human undergoing a lung transplant. Such use generally involves administration to an animal in need thereof one or more of the disclosed therapeutic delivery vehicles, in an amount and for a time sufficient to prevent, treat, lessen, or cure the disease, disorder, dysfunction, condition, or deficiency in the affected animal, and/or to ameliorate one or more symptoms thereof. In certain applications, the therapeutic compositions may be formulated to contain one or more anti -rejection, anti-inflammatory, or anti-microbial agents, or any combination thereof.

[0083] Typically, formulations of one or more of the drug delivery nanoparticles described herein will contain at least a chemotherapeutically-effective amount of a first active agent Preferably, the formulation may contain at least about 0.001% of each active ingredient, preferably at least about 0.01% of the active ingredient, although the percentage of the active ingredient(s) may, of course, be varied, and may conveniently be present in amounts from about 0.01 to about 90 weight % or volume %, or from about 0.1 to about 80 weight % or volume %, or more preferably, from about 0.2 to about 60 weight % or volume %, based upon the total formulation. Naturally, the amount of active compound(s) in each composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological t , route of administration, product shelf-life, as well as other pharmacological considerations will be contemplated by one of ordinary skill in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0084] Pharmaceutical formulations adapted for injectable administration include, but are not limited to, sterile aqueous solutions, dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions including, without limitation, one or more of those described in U.S. Patent No. 5,466,468 (specifically incorporated herein in its entirety by express reference thereto). In all cases, the form is preferably sterile, and is preferably fluid to the extent that easy syringability and/or ready administration to the patient is achievable. It is also preferably at least sufficiently stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as viruses, bacteria, fungi, and such like.

[0085] The carrier(s) can be a solvent or dispersion medium including, without limitation, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like, or a combination thereof), one or more vegetable oils, or any combination thereof, although additional pharmaceutically-acceptable components may be included.

[0086] Proper fluidity of the pharmaceutical formulations disclosed herein may be maintained, for example, by the use of a coating, such as, e.g., a lecithin, by the maintenance of the required particle size in the case of dispersion, by the use of a surfactant, or any combination of these techniques. The inhibition or prevention of the action of microorganisms can be brought about by one or more antibacterial or antifungal agents, for example, without limitation, a paraben, chlorobutanol, phenol, sorbic acid, thimerosal, or the like. In many cases, it will be preferable to include an isotonic agent, for example, without limitation, one or more sugars or sodium chloride, or any combination thereof. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example without limitation, aluminum monostearate, gelatin, or a combination thereof.

[0087] While systemic administration is contemplated to be effective in many embodiments as disclosed herein, it is also contemplated that formulations of the disclosed drug delivery compositions may be suitable for direct injection into one or more organs, tissues, or cell types in the body. Administration of the disclosed compositions may be conducted using suitable means, including those known to the one of ordinary skill in the relevant medical arts.

[0088] The pharmaceutical formulations as disclosed herein are not in any way limited to use only in humans, or even to primates, or mammals. In certain embodiments, the methods and compositions disclosed herein may be employed during organ transplantation in avian, amphibian, reptilian, or other animal species. In preferred embodiments, however, the compositions disclosed herein are preferably formulated for administration to a mammal, and in particular, to humans, undergoing a transplantation procedure, such as a lung transplant.

[0089] The compositions disclosed herein may also be provided in formulations that are acceptable for veterinary administration, including, without limitation, to selected livestock, exotic or domesticated animals, companion animals (including pets and such like), non-human primates, as well as zoological or otherwise captive specimens, and such like.

EXEMPLARY DEFINITIONS

[0090] In accordance with the present disclosure, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like, include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0092] The following references provide one of skill with a general definition of many of the terms used in this invention: Dictionary of Biochemistry and Molecular Biology, (2^nd Ed.) J. Stenesh (Ed.), Wiley-Interscience (1989); Dictionary of Microbiology and Molecular Biology (3^rd Ed.), P. Singleton and D. Sainsbury (Eds.), Wiley-Interscience (2007); Chambers Dictionary of Science and Technology (2^nd Ed.), P. Walker (Ed.), Chambers (2007); Glossary of Genetics (5^th Ed.), R. Rieger et al. (Eds.), Springer- Verlag (1991); and The HarperCollins Dictionary of Biology, W.G. Hale and J.P. Margham, (Eds.), HarperCollins (1991).

[0093] Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, and compositions are described herein. For purposes of the present disclosure, the following terms are defined below for sake of clarity and ease of reference:

[0094] In accordance with long standing patent law convention, the words "a" and "an," when used in this application, including the claims, denote "one or more."

[0095] The terms "about" and "approximately" as used herein, are interchangeable, and should generally be understood to refer to a range of numbers around a given number, as well as to all numbers in a recited range of numbers (e.g., "about 5 to 15" means "about 5 to about 15" unless otherwise stated). Moreover, all numerical ranges herein should be understood to include each whole integer within the range.

[0096] As used herein, the term "buffer" includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.

[0097] As used herein, the term "carrier" is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert (s), or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a therapeutic or diagnostic purpose, as applicable.

[0098] The term "decellularized organ" as used herein refers to an organ, or part of an organ from which the entire cellular and tissue content has been removed leaving behind a complex interstitial structure. Organs are composed of various specialized tissues. The specialized tissue structures of an organ are the parenchyma tissue, and they provide the specific function associated with the organ. Most organs also have a framework composed of unspecialized connective tissue which supports the parenchyma tissue. The process of decellularization removes the parenchyma tissue, leaving behind the three- dimensional interstitial structure of connective tissue, primarily composed of collagen. The interstitial structure has the same shape and size as the native organ, providing the supportive framework that allows cells to attach to, and grow on it. Decellularized organs can be rigid, or semi-rigid, having an ability to alter their shapes. Examples of decellularized organs include, but are not limited to the heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra.

[0099] As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term "DNA segment," are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.

[0100] The term "effective amount," as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.

[0101] As used herein, the terms "engineered" and "recombinant" cells are intended to refer to a cell into which an exogenous polynucleotide segment (such as DNA segment that leads to the transcription of a biologically active molecule) has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced exogenous DNA segment. Engineered cells are, therefore, cells that comprise at least one or more heterologous polynucleotide segments introduced through the hand of man.

[0102] As used herein, the term "epitope" refers to that portion of a given immunogenic substance that is the target of (i.e. , is bound by), an antibody or cell- surface receptor of a host immune system that has mounted an immune response to the given immunogenic substance as determined by any method known in the art. Further, an epitope may be defined as a portion of an immunogenic substance that elicits an antibody response or induces a T-cell response in an animal, as determined by any method available in the art (see, e.g., Geysen et al, 1984). An epitope can be a portion of any immunogenic substance, such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof. The term "epitope" may also be used interchangeably with "antigenic determinant" or "antigenic determinant site."

[0103] The term "for example" or "e.g. ," as used herein, is used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the specification.

[0104] As used herein, "heterologous" is defined in relation to a predetermined referenced DNA or amino acid sequence. For example, with respect to a structural gene sequence, a heterologous promoter is defined as a promoter that does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation. Likewise, a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements. [0105] As used herein, "homologous" means, when referring to polypeptides or polynucleotides, sequences that have the same essential structure, despite arising from different origins. Typically, homologous proteins are derived from closely related genetic sequences, or genes. By contrast, an "analogous" polypeptide is one that shares the same function with a polypeptide from a different species or organism, but has a significantly different form to accomplish that function. Analogous proteins typically derive from genes that are not closely related.

[0106] As used herein, the term "homology" refers to a degree of complementarity between two polynucleotide or polypeptide sequences. The word "identity" may substitute for the word "homology" when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence. Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.

[0107] The terms "identical" or percent "identity," in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0108] As used herein, the phrase "in need of treatment" refers to a judgment made by a caregiver such as a physician or veterinarian that a patient requires (or will benefit in one or more ways) from treatment. Such judgment may made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient is ill as the result of a disease state that is treatable by one or more compound or pharmaceutical compositions such as those set forth herein.

[0109] The phrases "isolated" or "biologically pure" refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state. Thus, isolated polynucleotides in accordance with the invention preferably do not contain materials normally associated with those polynucleotides in their natural, or in situ, environment.

[0110] The term "isolated organ" as used herein refers to an organ that has been removed from a mammal. Suitable mammals include humans, primates, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep. The term "isolated organ" also includes an organ removed from the subject requiring an artificial reconstructed organ. Suitable organs can be any organ, or part of organ, required for replacement in a subject. Examples include but are not limited to the heart, lung, kidney, liver, pancreas, spleen, bladder, ureter and urethra.

[0111] An organ, or a part of an organ, can be isolated from the subject requiring an artificial reconstructed organ. For example, a diseased organ in a subject can be removed and decellularized, as long as the disease effects the parenchyma tissue of the organ, but does not harm the connective tissue, e.g., tissue necrosis. The diseased organ can be removed from the subject and decellularized. The decellularized organ, or a part of the organ, can be used as a three-dimensional scaffold to reconstruct an artificial organ. An allogenic artificial organ can be reconstructed using the subject's own decellularized organ as a scaffold and using a population of cells derived from the subject's own tissue. For example, cells populations derived from the subject's skin, liver, pancreas, arteries, veins, umbilical cord, and placental tissues.

[0112] A xenogenic artificial organ can be reconstructed using the subject's own decellularized organ as a scaffold, and using cell populations derived from a mammalian species that are different from the subject. For example the different cell populations can be derived from mammals such as primates, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep.

[0113] An organ, or part of an organ, can also be derived from a human cadaver, or from mammalian species that are different from the subject, such as organs from primates, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep. Standard methods for isolation of a target organ are well known to the skilled artisan and can be used to isolate the organ.

[0114] As used herein, the term "kit" may be used to describe variations of the portable, self-contained enclosure that includes at least one set of reagents, components, or pharmaceutically-formulated compositions to conduct one or more of the assay methods of the present disclosure. Optionally, such kit may include one or more sets of instructions for use of the enclosed reagents, such as, for example, in a laboratory or clinical application.

[0115] "Link" or "join" refers to any method known in the art for functionally connecting one or more proteins, peptides, nucleic acids, or polynucleotides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like. [0116] The terms "local administration" or "local delivery," in reference to delivery of a composition, formulation, or device of the invention, refer to delivery that does not rely upon transport of the agent to its intended target tissue via the vascular or lymphatic system from a site of administration that is remote from the intended target tissue. The agent is delivered directly to its intended target tissue or in the vicinity thereof, e.g. by injection or implantation. It will be appreciated that a small amount of the delivered agent may enter the vascular system and may ultimately reach the target tissue via the vascular system.

[0117] As used herein, "mammal" refers to the class of warm-blooded vertebrate animals that have, in the female, milk- secreting organs for feeding the young. Mammals include without limitation humans, apes, many four-legged animals, whales, dolphins, and bats. A human is a preferred mammal for purposes of the invention.

[0118] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally-occurring. As used herein, laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally-occurring animals.

[0119] As used herein, the term "nucleic acid" includes one or more types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites). The term "nucleic acid," as used herein, also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like. "Nucleic acids" include single- and double- stranded DNA, as well as single- and double-stranded RNA. Exemplary nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.

[0120] The term "operably linked," as used herein, refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.

[0121] As used herein, the term "patient" (also interchangeably referred to as "recipient" "host" or "subject") refers to any host that can serve as a recipient for one or more of the vascular access devices as discussed herein. In certain aspects, the recipient will be a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being). In certain embodiments, a "patient" refers to any animal host, including but not limited to, human and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals or birds, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner.

[0122] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that preferably do not produce an allergic or similar untoward reaction when administered to a mammal, and in particular, when administered to a human. As used herein, "pharmaceutically acceptable salt" refers to a salt that preferably retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, without limitation, acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like); and salts formed with organic acids including, without limitation, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; salts formed with an organic cation formed from Ν,Ν' dibenzylethylenediamine or ethylenediamine; and combinations thereof.

[0123] As used herein, "pharmaceutically-acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, but are not limited to, acid-addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; salts formed with an organic cation formed from Ν,Ν'-dibenzylethylenediamine or ethylenediamine; and combinations thereof.

[0124] As used herein, the term "plasmid" or "vector" refers to a genetic construct that is composed of genetic material (i.e., nucleic acids). Typically, a plasmid or a vector contains an origin of replication that is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells including the plasmid. Plasmids and vectors of the present invention may include one or more genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in a suitable expression cells. In addition, the plasmid or vector may include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments that are obtained from or derived from one or more natural and/or artificial sources.

[0125] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to "peptide," "dipeptide," "tripeptide," "protein," "enzyme," "amino acid chain," and "contiguous amino acid sequence" are all encompassed within the definition of a "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone one or more post- translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post- translation processing, or modification by inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures. For example, one-letter and three-letter abbreviations are widely employed to describe amino acids: Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; lie), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys). Amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for L- amino acid residues provided the desired properties of the polypeptide be retained.

[0126] As used herein, the terms "prevent," "preventing," "prevention," "suppress," "suppressing," and "suppression" as used herein refer to administering a compound either alone or as contained in a pharmaceutical composition prior to the onset of clinical symptoms of a disease state so as to prevent any symptom, aspect or characteristic of the disease state. Such preventing and suppressing need not be absolute to be deemed medically useful.

[0127] The term "promoter," as used herein refers to a region or regions of a nucleic acid sequence that regulates transcription.

[0128] "Protein" is used herein interchangeably with "peptide" and "polypeptide," and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject. The term "polypeptide" is preferably intended to refer to any amino acid chain length, including those of short peptides from about two to about 20 amino acid residues in length, oligopeptides from about 10 to about 100 amino acid residues in length, and longer polypeptides including from about 100 amino acid residues or more in length. Furthermore, the term is also intended to include enzymes, i.e., functional biomolecules including at least one amino acid polymer. Polypeptides and proteins of the present invention also include polypeptides and proteins that are or have been post-translationally-modified, and include any sugar or other derivative(s) or conjugate(s) added to the backbone amino acid chain.

[0129] "Purified," as used herein, means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure. A compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e. , is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. A partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found, e.g., cellular material such as cellular proteins and/or nucleic acids.

[0130] A compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e., is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. A partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found(<?.g., cellular material such as cellular proteins, peptides, nucleic acids, etc.).

[0131] The term "recombinant" indicates that the material (e.g., a polynucleotide or a polypeptide) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within or removed from, its natural environment, or native state. Specifically, e.g., a promoter sequence is "recombinant" when it is produced by the expression of a nucleic acid segment engineered by the hand of man. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein which is produced by expression of a recombinant nucleic acid; and a "recombinant virus," e.g., a recombinant AAV virus, is produced by the expression of a recombinant nucleic acid.

[0132] The term "regulatory element," as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.

[0133] The term "RNA segment" refers to an RNA molecule that has been isolated free of total cellular RNA of a particular species. Therefore, RNA segments can refer to one or more RNA segments (either of native or synthetic origin) that have been isolated away from, or purified free from, other RNAs. Included within the term "RNA segment," are RNA segments and smaller fragments of such segments.

[0134] The term "sequence," when referring to amino acids, relates to all or a portion of the linear N-terminal-to-C-terminal order of amino acids within a given amino acid chain, e.g. , polypeptide or protein; "subsequence" means any consecutive stretch of amino acids within a sequence, e.g., at least 3 consecutive amino acids within a given protein or polypeptide sequence. With reference to nucleotide and polynucleotide chains, "sequence" and "subsequence" have similar meanings relating to the 5'-to-3' order of nucleotides.

[0135] The term "biologically functional equivalent" is well understood in the art, and is further defined in detail herein. Accordingly, sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the invention.

[0136] The term "substantially corresponds to," "substantially homologous," or "substantial identity," as used herein, denote a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent sequence identity, and more preferably, at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More preferably still, highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to which it was compared.

[0137] The percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, in the case of sequence homology of two or more polynucleotide sequences, the reference sequence will typically comprise at least about 18-25 nucleotides, more typically at least about 26 to 35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or even 100 or so nucleotides.

[0138] When highly-homologous fragments are desired, the extent of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more of the sequence comparison algorithms well-known to those of skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988).

[0139] "Sequential administration" of two or more agents refers to administration of two or more agents to a subject such that the agents are not present together in the subject's body at greater than de minimis concentrations. Administration of the agents may, but need not, alternate. Each agent may be administered multiple times. [0140] "Significant sequence homology" as applied to an amino acid sequence means that the sequence displays at least approximately 20% identical or conservatively replaced amino acids, preferably at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60% identical or conservatively replaced amino acids, desirably at least approximately 70% identical or conservatively replaced amino acids, more desirably at least approximately 80% identical or conservatively replaced amino acids, and most desirably at least approximately 90% amino acid identical or conservatively replaced amino acids relative to a reference sequence. When two or more sequences are compared, any of them may be considered the reference sequence. % identity can be calculated using a FASTA or BLASTP algorithm, using default parameters. A PAM250 or BLOSUM62 matrix may be used. For purposes of calculating % identical or conservatively replaced residues, a conservatively replaced residue is considered identical to the residue it replaces. Conservative replacements may be defined in accordance with Stryer, L, Biochemistry, 3rd ed., 1988, according to which amino acids in the following groups possess similar features with respect to side chain properties such as charge, hydrophobicity, aromaticity, etc. (1) Aliphatic side chains: G, A, V, L, I; (2) Aromatic side chains: F, Y, W; (3) Sulfur-containing side chains: C, M; (4) Aliphatic hydroxyl side chains: S, T; (5) Basic side chains: K, R, H; (6) Acidic amino acids: D, E, N, Q; and (7) Cyclic aliphatic side chain: P.

[0141] The term "subject," as used herein, describes an organism, including mammals such as primates, to which treatment with the compositions according to the present disclosure can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, non-human primates such as apes; chimpanzees; monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.

[0142] "Substantial sequence homology" as applied to a sequence means that the sequence displays at least approximately 60% identity, desirably at least approximately 70% identity, more desirably at least approximately 80% identity, and most desirably at least approximately 90% identity relative to a reference sequence. When two or more sequences are compared, any of them may be considered the reference sequence. % identity can be calculated using a FASTA, BLASTN, or BLASTP algorithm, depending on whether amino acid or nucleotide sequences are being compared. Default parameters may be used, and in exemplary embodiments, a PAM250 and/or BLOSUM62 matrix or such like may be employed in the practice of the invention.

[0143] A "sustained release formulation" is a composition of matter that comprises a therapeutic agent as one of its components and further comprises one or more additional components, elements, or structures effective to provide sustained release of the therapeutic agent, optionally in part because of the physical structure of the formulation. Sustained release is release or delivery that occurs either continuously or intermittently over an extended period, e.g., at least several days, at least several weeks, at least several months, at least several years, or even longer, depending upon the particular formulation employed.

[0144] "Suitable standard hybridization conditions" for the present disclosure include, for example, hybridization in 50% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg/ml of denatured salmon sperm DNA at 42°C for 16 hr followed by 1 hr sequential washes with O.lx SSC, 0.1% SDS solution at 60°C to remove the desired amount of background signal. Lower stringency hybridization conditions for the present disclosure include, for example, hybridization in 35% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg/ml denatured salmon sperm DNA or E. coli DNA at 42°C for 16 h followed by sequential washes with 0.8x SSC, 0.1% SDS at 55°C. Those of skill in the art will recognize that conditions can be readily adjusted to obtain the desired level of stringency.

[0145] As used herein, the term "structural gene" is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.

[0146] Naturally, the present disclosure also encompasses nucleic acid segments that are complementary, essentially complementary, and/or substantially complementary to at least one or more of the specific nucleotide sequences specifically set forth herein. Nucleic acid sequences that are "complementary" are those that are capable of base- pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to one or more of the specific nucleic acid segments disclosed herein under relatively stringent conditions such as those described immediately above.

[0147] As described above, the probes and primers of the present disclosure may be of any length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc. , an algorithm defining all probes or primers contained within a given sequence can be proposed:

n to n + y,

where n is an integer from 1 to the last number of the sequence, and y is the length of the probe or primer minus one, where n + y does not exceed the last number of the sequence.

[0148] Thus, for a 25 -basepair probe or primer (i.e., a "25 mer"), the collection of probes or primers correspond to bases 1 to 25, bases 2 to 26, bases 3 to 27, bases 4 to 28, and so on over the entire length of the sequence. Similarly, for a 35-basepair probe or primer (i.e., a "35-mer), exemplary primer or probe sequence include, without limitation, sequences corresponding to bases 1 to 35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over the entire length of the sequence. Likewise, for 40-mers, such probes or primers may correspond to the nucleotides from the first basepair to bp 40, from the second bp of the sequence to bp 41, from the third bp to bp 42, and so forth, while for 50-mers, such probes or primers may correspond to a nucleotide sequence extending from bp 1 to bp 50, from bp 2 to bp 51, from bp 3 to bp 52, from bp 4 to bp 53, and so forth.

[0149] The term "substantially complementary," when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an mRNA encoding the selected sequence. As such, typically the sequences will be highly complementary to the mRNA "target" sequence, and will have no more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 or so base mismatches throughout the complementary portion of the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e., be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product. [0150] Substantially complementary nucleic acid sequences will be greater than about 80 percent complementary (or "% exact-match") to a corresponding nucleic acid target sequence to which the nucleic acid specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the invention, and in such instances, the nucleic acid sequences will be greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including about 96%, about 97%, about 98%, about 99%, and even about 100% exact match complementary to all or a portion of the target sequence to which the designed nucleic acid specifically binds.

[0151] Percent similarity or percent complementary of any of the disclosed nucleic acid sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0152] The term "therapeutically-practical period" means the period of time that is necessary for one or more active agents to be therapeutically effective. The term "therapeutically-effective" refers to reduction in severity and/or frequency of one or more symptoms, elimination of one or more symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and the improvement or a remediation of damage.

[0153] A "therapeutic agent" may be any physiologically or pharmacologically active substance that may produce a desired biological effect in a targeted site in a subject. The therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, a cytokine, a cytotoxic agent, a nucleolytic compound, a radioactive isotope, a receptor, and a pro-drug activating enzyme, which may be naturally-occurring, or produced by synthetic or recombinant methods, or any combination thereof. Drugs that are affected by classical multidrug resistance, such as the vinca alkaloids (e.g., vinblastine and vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) and microtubule stabilizing drugs (e.g., paclitaxel) may have particular utility as the therapeutic agent. Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. A cancer chemotherapy agent may be a preferred therapeutic agent. For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician 's Desk Reference and to Goodman and Gilman's "Pharmacological Basis of Therapeutics" tenth edition, Hardman et al. (Eds.) (2001).

[0154] "Transcriptional regulatory element" refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences. A transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.

[0155] As used herein, a "transcription factor recognition site" and a "transcription factor binding site" refer to a polynucleotide sequence(s) or sequence motif(s), which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding. Typically, transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted based on known consensus sequence motifs, or by other methods known to those of ordinary skill in the art.

[0156] "Transcriptional unit" refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first ds-acting promoter sequence and optionally linked operably to one or more other cw-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as well as any additional cis sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc. As used herein, the term "transformed cell" is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell.

[0157] As used herein, the term "transformation" is intended to generally describe a process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast in which the exogenous polynucleotide is incorporated into at least a first chromosome or is capable of autonomous replication within the transformed host cell. Transfection, electroporation, and "naked" nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.

[0158] "Treating" or "treatment of as used herein, refers to providing any type of medical or surgical management to a subject. Treating can include, but is not limited to, administering a composition comprising a therapeutic agent to a subject. "Treating" includes any administration or application of a compound or composition of the invention to a subject for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder, or condition. In certain aspects, the compositions of the present invention may also be administered prophylactically, i.e. , before development of any symptom or manifestation of the condition, where such prophylaxis is warranted. Typically, in such cases, the subject will be one that has been diagnosed for being "at risk" of developing such a disease or disorder, either as a result of familial history, medical record, or the completion of one or more diagnostic or prognostic tests indicative of a propensity for subsequently developing such a disease or disorder.

[0159] The term "vector," as used herein, refers to a nucleic acid molecule (typically comprised of DNA) capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment. A plasmid, cosmid, or a virus is an exemplary vector.

[0160] In certain embodiments, it will be advantageous to employ one or more nucleic acid segments of the present disclosure in combination with an appropriate detectable marker (i.e., a "label,"), such as in the case of employing labeled polynucleotide probes in determining the presence of a given target sequence in a hybridization assay. A wide variety of appropriate indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, without limitation, fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, etc., which are capable of being detected in a suitable assay. In particular embodiments, one may also employ one or more fluorescent labels or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally less-desirable reagents. In the case of enzyme tags, colorimetric, chromogenic, or fluorigenic indicator substrates are known that can be employed to provide a method for detecting the sample that is visible to the human eye, or by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, and the like, to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences. In the case of so-called "multiplexing" assays, where two or more labeled probes are detected either simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (for example, an emission and/or excitation spectral maximum), which also labeled a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., discreet or discernible from the first label. The use of multiplexing assays, particularly in the context of genetic amplification/detection protocols are well-known to those of ordinary skill in the molecular genetic arts.

[0161] The section headings used throughout are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application (including, but not limited to, patents, patent applications, articles, books, and treatises) are expressly incorporated herein in their entirety by express reference thereto. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

EXAMPLES

[0162] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 - APPARATUS FOR DECELLULARIZATION OF ORGAN TISSUES

[0163] Photographs of an exemplary bioreactor are shown in FIG. 2 and FIG. 3, with and without an attached organ, respectively. The techniques tested were all concerning a perfusion and immersion decellularization approach, since lungs were eased down in the bioreactor connected with the tubes as in the scheme, i.e., a tube connected to the pulmonary artery (PA) and one to the trachea, and then completely immersed in the proper solution until the bioreactor was filled up. The standard configuration considers the presence of a tube having one end connected with the PA passing through a pump and the other end left free in the bioreactor, another tube connected in the same way of the one just described for the trachea and finally a tube with both the end terminating in the bioreactor and a pump connected to allow for recycle of the solution.

[0164] Before being connected to the proper tube system lungs were ventilated with a Hamilton-C2 (Hamilton Medical, Inc. clinical ventilator to measure their mechanical properties such as compliance and resistance; two modes of ventilation were applied: one in constant volume (that means the volume of air pushed into the lung per each cycle of breathing is kept constant, see FIG. 4A and FIG. 4B for screen-shots of the ventilator monitor) and another in constant pressure (meaning that the pressure during inhalation is kept to a constant value, see (Sawada et al, 2008for the setting screen-shot).

[0165] The bubbling technique developed during the decellularization of lung 6 (FIG. 7) and 7 (FIG. 8) and consol- idated for lung 8 was designed entirely from the beginning, a paper regarding an industrial technique employed for surfactant removal form air/water interface (Arand et al, 1992) was used as a spark to develop the method described in the definitive decellularization protocol (Sawada et al, 2008). As it is possible to grasp in the following picture (Sawada et al, 2008) the techniques makes use of air bubbles pushed from the bottom of the bioreactor, filled with deionized water, to force the SDS molecules still present in the rinsing solution to self assembly and move towards the liquid/air surface; here they accumulate and form a layer of bubbles which created a pressure gradient which guides them towards the tube connected to the drain where they encounter the less resistance. Thus bubbles are force to leave the reactor as they form. After a while the air compressor used to creates the bubbles is stopped and fresh deionized water is pushed in the bioreactor from the bottom of the reactor throughout the same tubes used for the air; the fresh water inserted pushed the liquid just beneath the liquid/air interface towards the drain port where it leaves the bioreactor. Thus fresh water substitutes the old one which is thought to contain more SDS. It is evident, also from the results presented below, that using deionized water to cover the lung helps to extract SDS form the inside of the organ by diffusion and it is also clear that this phenomenon is helped by the bubbles that gently it the lung anchored to the bioreactor.

[0166] The studies of the mechanical properties of the lung were performed with a ventilator as already mentioned in the section of materials and methods. Lungs were ventilated in 2 different modes: the APVcmv, that is the constant volume mode and the PCV+, that is, instead the pressure constant mode. As expected the values of compliance and resistance after the decellularization changed quite significantly with respect to the values before the decellularization; this is of course due to tha fact that ECM scaffolds has been slightly modified by the solution employed and also the fact that cells like the AEC II were removed is considered to be an important factor for this phenomenon: AEC II cells, in fact, secrete surfactants that usually help the lung to be more compliant in the inspiration phase.

[0167] For each mode 3 pictures of the ventilator screen were taken, one to catch the curve of volume (given in ml) versus pressure (given in cmH2 0), another to get the curve flow (given in Ijmin) versus pressure and the last one the curve volume versus flow. So for every lung, 6 pictures were collected and studied. As it is easy to see in these figures, the general trend was an increase of the value of the resistance when going from a normal lung to an acellular one, and a decrease, of course, of the compliance.

EXAMPLE 2 -DECELLULARIZATION OF ORGAN TISSUES

[0168] A first exemplary protocol utilized for the decellularization of organ tissues is reported below; it involves only the exploitation of an SDS solution in deionized water for the entire process:

DECELLULARIZATION PROCESS USING ONLY 2% SDS

[0169] Connect pulmonary artery (PA) and trachea of lungs to separate cannulas.

[0170] Immerse entire lungs in 2% SDS and pump 2% SDS through trachea (100 niL/min for 5 min into lungs, 100 niL/min for 5 min out of lung) and PA (100 niL/min continuously). Recycle the SDS solution in the bioreactor at a rate of 300 mLVmin. [0171] Change out all SDS to fresh 2% SDS after the first day and then every two days after that. Decellularization is considered complete when there are no longer any visible pink areas on lungs.

[0172] Since the results obtained were not satisfying as expected, as we will discuss in the proper section, the protocol was changed and different concentrations of SDS in solution were tested, but the decellularized lungs looked too damaged and spoiled to be taken as a good technique. Although improvements were achieved by employing only SDS solutions, the first goal of obtaining a perfect AC lung showing good ECM maintenance for further decellularization was up to that moment not reached. To improve this process keeping SDS as solvent for the decellularization, a new solution was added to the protocol; a solution of 0.0023 of dextrose in deionized water was used indeed to prepare lungs to be treated with SDS solution again, and the main difference was that this decellularization procedure would have last for just a couple of hours instead of days. The results ended up to be better with this new approach and also with the employment of non-frozen lungs, i.e., lungs harvested and immediately decellularized.

DECELLULARIZATION PROCESS USING 0.002% DEXTROSE AND 2% SDS

[0173] Ventilate the lung in constant volume mode (APV_cmv mode: V_t = 300 mL, PEEP/CPAP=5 cm H₂0, Oxygen = 100%) and constant pressure mode (PCV+ mode: P_control = 20 cm H₂0, PEEP/CPAP= 5 cm H₂0, oxygen = 100%).

[0174] Take pictures of the lung during ventilations, every time decellularization solution samples are collected, before and after every solution change and every time there is something unusual going on.

[0175] Connect pulmonary artery (PA) to the tube and connect the trachea to the special home-made connector that has one tube connect to a pump and another that goes to one of the free port at the top of the bioreactor, makes a loop and goes back again into the bioreactor, draining the eventual liquid into the bioreactor chamber.

[0176] Immerse entire lungs in 0.002% dextrose and pump 0.002% dextrose through PA (100 mL/min continuously) and through trachea (100 mU min down it for 2 min and then stop the pump for other 2 min).

[0177] Use third pump to circulate the solution by pulling from the bottom of the tank and filling at the middle of the bioreactor at a rate of 350 mL/min. [0178] After 24 hrs remove the 0.002% dextrose solution and substitute that with new 0.002% dextrose and run it for another 24 hrs.

[0179] After 24 hrs repeat step 4.

[0180] After 24 hrs, thus at the beginning of day 4 of the decellularization process, remove the 0.002% dextrose solution and substitute that with 2% SDS solution. Every hour remove Ά of the amount of the solution and refill the Bioreactor with fresh 0.2% to keep the solution changed. During this step keep the bioreactor horizontally and do not perfuse down to the trachea leaving all the tubes hooked up as during dextrose treatment.

[0181] After 4 hrs of 2% SDS treatment, stop the process. If lung appears pink in color, continue with 2% SDS treatment for additional 1-3 hrs.

[0182] Decellularization is considered complete when there are no longer any visible pink regions of the lungs.

[0183] Rinse the lung using the following protocol for 3 hrs: LUNG RINSE USING MILLIQ WATER

[0184] Connect pulmonary artery (PA) and trachea as done for decellularization process (leave the lung "hooked-up" to the bioreactor if the wash is to be performed right after decellularization process).

[0185] Leave the lung lying on the bigger wall of the bioreactor so that is not subjected to further pressure due to gravity force and pump fresh MilliQ water through the PA (100 mL/min continuously) and through the trachea (100 mL/min for 2 min, and after that stop the pump for other 2 min leaving the third ends of the T-connector open for drainage, (perform this as a cycle).

[0186] Wash the lung from the top with MilliQ water and a third pump is used to push waste liquid from the bottom of the bioreactor to the sink.

[0187] Rinse the lung using the following protocol until all the bubbles formed due to the presence of SOS are gone:

BUBBLING/OVERFLOWING TECHNIQUES FOR REMOVING SDS FROM LUNGS

[0188] Leave the lung connected to the Bioreactor as it was before for the decellularization process. Then add a pump/air compressor or use the one that before was employed for the recycling and make it push air from outside to the bottom of the reactor, thus having bubbles forming on the surface of the solution. [0189] Connect a tube to one of the top port of the bioreactor thus that when too much bubbling increases the pressure in the container bubbles are forced to take the only free tube that must end in the drain.

[0190] Fill up the bioreactor with the proper solution (that may be MilliQ water, 0.002% Dextrose or PBS depending on what was planned) until the bioreactor is almost completely full of solution.

[0191] Check that all the sealing parts are closed and not leaky.

[0192] Run the others pump as done for the decellularization protocol.

[0193] Once the time of bubbling is finished, use the pump employed to create bubbles or a free pump to push liquid to the bottom of the reactor, so that the liquid level will increase leading the first level of that just above and below the liquid surface to leave the bioreactor by means of the tube connected for the overflowing of the bubbles.

[0194] At least once every day, during this process, collect samples of the liquid at the bottom of the bioreactor and subject that to methylene blue procedure to check SDS concentration and see if it is going down, otherwise interrupt the process since that would mean it is not efficient.

[0195] If the bubbles are still there after too much time repeat step 8, otherwise follow step 10.

[0196] Rinse the lung as done in step 8 but this time use for the first 2 hrs a solution made of either 10% ethanol or hydrogen peroxide then switch again to MilliQ water to remove the disinfectant that may damage further the scaffold.

[0197] Ventilate it in constant pressure and constant volume mode using the same parameters as described in step 1.

[0198] Collect samples (4 x 1 mL) every hour while treating with 0.002% dextrose for the first 6 hrs since the change of the solution.

[0199] Collect samples (4 x 1 mL) every time before changing the solution.

[0200] (Add to 20 liters of 0.002% dextrose solution 100 mL of solution containing:

90 μg/mL of streptomycin, 50 U/mL of penicillin, and 25 μg/mL of amphotenicin B).

[0201] STORE the AC lung in IX PBS solution with antibiotics and antimycotics at 3°C.

[0202] Collect tissue samples following the relevant protocol.

[0203] Perform BCA protein Assay of the decellularization solution samples following the relevant protocol. [0204] Perform SOS quantification in the liquid samples following the relevant protocol.

[0205] Homogenize tissue samples and perform SOS concentration quantification and BCA protein assay with the liquid obtained.

[0206] As described in the protocol just shown, every hour for the first 6 hrs since the substitution of the decellularization solution and before the substitution, but not during the wash, samples of the solution were collected and stored for future protein amount evaluation by means of standard BCA Protein Essay kit (Thermo Fisher Scientific, Inc.). After the decellularization process, lungs were sectioned in order to collect tissue samples fixed with Formalin, OCT compound or directly snap-frozen in liquid nitrogen; these samples contained different kinds of tissue, more exactly pieces of bronchioles, veins and parenchyma were picked from every lobe; they were then stored at 80°C for later histology and protein quantification assay. In fact, some of the parenchyma samples were homogenized through a proper technique, and then the amount of protein was studied again with BCA Protein Essay kit. Other samples were, instead, stained for immunofluorescence studies, during this first experience we were able to stain for collagen I and III only, leaving staining of other protein for future and improved analysis; to look at those kinds of collagen, antibodies of type IgG made for rabbit and pig collagen I and III (produced by AbD Serotec, Bio-Rad Laboratories, Inc.) were used as primary antibodies, while goat anti-rabbit IgG H& L (FITC) (Abeam, Inc.) were used as secondary antibodies.

[0207] An important and innovative technique that was introduced during this work in the field of decellularization thorough SDS solution is its quantification using a procedure involving reagent containing methylene blue (Arand et al., 1992).

EXAMPLE 3 - SDS REMOVAL AND BUBBLING TECHNIQUE DEVELOPMENT

[0208] Starting from lung #2, it began to be evident the necessity to remove SDS from lungs after the decellularization process, so that is why several attempts were conducted before arriving at the final "bubbling" technique. Lung #3 was washed with deionized water for hours; the washing technique used for that entailed the perfusion of deionized water down to the pulmonary artery and the trachea while in the meantime other purified water was poured directly on top of it, keeping a pump to drain the liquid accumulated in the bioreactor. [0209] During these studies it was difficult to establish whether the SDS had been removed properly, since there was no method to quantify the SDS present; at this point a chemist of our group came out with the technique previously reported (Arand et al, 1992). This quick and inexpensive technique allowed for the quantification of the concentration of SDS in the rinsing solution, representing a turning point for the development of the following process of wash. Before getting to the bubbling technique several attempts were conducted as sated above; among with water, also IX PBS and 10% Ethanol were tried. Ethanol was tried since it would also give a good method for disinfecting the organ: unfortunately for safety reason it was not possible to employ 70% concentration which is known to be the most effective one for disinfecting, but quick trails of piece of acellular tissue immersed in a special growing media for bacteria did not show any evidence of rapid growing of bacterial colony, proving that also 10% ethanol could be quite effective in eliminating bacteria in the organ. It must also be noticed that during the process of the dextrose contains antibacterials and antimicotics, and that SDS may help to lyse bacterial membranes.

[0210] Although these several tries improved the previous situation, the SDS content in the rinsing solution and in the acellular tissue of the scaffold was still high (FIG. 5). Pieces of acellular tissue were dried and homogenized in deionized water, and then analyzed with the usual SDS quantification technique to account for its presence compared to the mass of the sample; this allowed a comparison between different techniques to determine which one would better achieve the desired goal.

[0211] The bubbling technique developed during the decellularization of lung 6 and 7 and consolidated for lung #8 (FIG. 9) was designed entirely from the beginning, a paper regarding an industrial technique employed for surfactant removal form air/water interface (Kou and Saylor, 2008) was used as a spark to develop the method described in the definitive decellularization protocol. As can be seen in FIG. 12, these techniques makes use of air bubbles pushed from the bottom of the bioreactor, filled with deionized water, to force the SDS molecules still present in the rinsing solution to self-assembly, and move towards the liquid/air surface; here they accumulate and form a layer of bubbles which created a pressure gradient which guides them towards the tube connected to the drain where they encounter the less resistance. Thus bubbles are force to leave the reactor as they form. After a while the air compressor used to creates the bubbles is stopped and fresh deionized water is pushed in the bioreactor from the bottom of the reactor throughout the same tubes used for the air; the fresh water inserted pushed the liquid just beneath the liquid/air interface towards the drain port where it leaves the bioreactor. Thus, the fresh water substitutes for the old one which is thought to contain more SDS. It is evident, also from the results presented below, that using deionized water to cover the lung helps to extract SDS form the inside of the organ by diffusion and it is also clear that this phenomenon is helped by the bubbles that gently it the lung anchored to the bioreactor.

[0212] To prove the efficiency of this method different analysis were conducted; samples of rinsing solution were collected at different moments, to be more precise, samples were collected before the wash (denoted Baseline) after 2 hrs since the beginning of the wash (termed "2 hrs"), after 24 hrs and after 27 hrs that is just before the interruption of the process; samples of the bubbles drained and of the overflow of the solution were analyzed too and plotted on a graph (FIG. 13, FIG. 14A, FIG. 14B, and FIG. 14C).

[0213] It is interesting to notice that the concentration of the overflowed solution and of the bubbles drained are the highest ones as it is easy to see on the relative plot (FIG. 14A). From the plots shown, it was clear that this technique was able to provide an SDS concentration of the order of thousandths in percentage (the average of 5 lungs was 0.0083) starting from a concentration of 23.

[0214] The most interesting results were, above all, the amount of SDS left in the acellular matrix; indeed this is the real goal that it is require to achieve. Parenchyma decellularized tissue samples were dried, homogenized in deionized water and then analyzed to find out the ration between the mass of SDS present and the mass of the sample. This allowed comparison of the results from different lungs and proved the efficacy of the technique (FIG. 14B). Comparing the ratios obtained from the five lungs washed with the "bubbling" technique, and the ones for the lungs washed simply by perfusion of deionized water gave the proof of that as shown in FIG. 14C.

[0215] The previous pictures showed pretty well how the bubbling technique works better compared with any other else. This simple but really effective method may be transferred to the decellularization of different organs that make use of SDS as principle decellularizing solution and solve the big issue of its removal from acellular tissue.

PROTEIN AMOUNT EVALUATION

[0216] Understanding the amount of protein that are released from the organ during the decellularization process is really important; of course it is difficult to define a threshold that allows us to say whether the amount of protein come off the lung is high or not, but it is still a study that can not miss in this part of the research, since every aspect should be taken into consideration. Moreover the BCA Protein Essay (Pierce Biotechnology, Inc.) that was used to conduct these analysis is quite cheap and easily affordable.

[0217] The first study carried out with the above mentioned kit was the evaluation of the protein dissolved in the 0.002% Dextrose solution used during the first step of the decellularization process; as it is easy to catch from the graph (FIG. 16), after a couple of hours since the starting of the process or the change of the solution with a fresh one, the protein amount looks to assume a kind of stationary behavior as if the curve was getting into a saturation zone; this aspect was of fundamental importance especially during the developing of the protocol because it lead us to think about the necessity to keep changing the solutions with fresh ones in order to avoid a saturation of those and thus a loss of the decellularization efficiency; this saturating trend was present also while using SDS and this is the reason why the final protocol developed makes use of an almost continuous chang- ing of SDS solution, thus allowing for a more efficient and shorter decellularization activity.

[0218] Acellular parenchyma tissue samples were also homogenized and analyzed to look for the protein content left in the scaffold. This technique was also applied to control lung tissue so that it was possible to compare the results and see the difference between a normal tissue and an acellular one (FIG. 17A and FIG. 17B). As expected the amount of protein in the 5 lungs studied was much lower compared to the one of the controls, meaning that the decellularization may act not only on cells, which for sure contain protein, but also on the protein content of ECM. This is, actually, a question that needs to be investigated further: knowing whether this lowering of protein content is due to cell detachment from the scaffold or if it also a question of ECM protein removal could bring tremendous improvements in the decellularization protocols and also in understanding better the protein composition of organ scaffolds.

[0219] A pro-inflammatory environment, specifically with the presence of INF-γ and TNF-k is needed to activate the immunosuppressive effects of MSCs (Macchiarini et al, 2008; Crapo et al , 2011). Activated MSCs then exert immune-modulatory effects by the secretion of soluble factors and by cell-cell contact dependent interactions upon activation with pro-inflammatory cytokines. MSCs have been reported to secrete variety of products that aid in control of immune responses and lung responses to injury such as transforming growth factor β (TGF-β), interleukin 10 (IL-10), prostaglandin E2 (PGE2), and programmed death ligand 1 (PDL1) (Crapo et al, 2011; Song and Ott, 2011; Cortiella et al, 2010; Hopkinson et al, 2008; and Funamoto et al, 2010). MSCs play a role in immunomodulation and control of immune responses (Crapo et al, 2011; Song and Ott, 2011; Cortiella et al, 2010; Hopkinson et al, 2008; and Funamoto et al, 2010), and recently data has been generated that supports a role for MSCs in the regulation of tumor development in pancreatic cancer (Phillips et al, 2010). Preliminary data indicates the ability to identify and isolate MSCs from lung or peripheral blood. Human MSCs can be identified by the expression of a specific set of cell surface molecules such as CD105, CD90, CD29 and CXCR4 co-expression which can be used to evaluate the presence of MSCs in normal as well as injured lung environments. In a recently published study we isolated and characterized a series of human peripheral blood derived (HPBD) MSC cell subsets which we examined for ability to proliferate, differentiate into specific cell lineages, produce immunomodulatory or protective factors and migrate in vitro and in vivo (Sawada et al, 2008). Recently published work has shown that MSCs produce factors that modulate the immune response, reduce inflammation and even reduce the induction of apoptosis in tissues following injury (Sawada et al, 2008). MSC subsets have been induced to differentiate into neuronal lineages following priming with retinoic acid (RA) and neural growth factors (Sawada et al, 2008). These data support the important critical role played by MSCs in the control of damage in the lung and produce lineages of cells that may be required in the development of engineered lung tissue. It has been shown that RA-primed MSCs were able to ameliorate some of the damage and cognitive defects related to traumatic brain injury following transplantation into brains of TBI rats which supports the concept of production of specific MSC subpopulations for development of a stem cell based therapy. Changes that occur in endogenous populations of MSC responses could influence lung function and lung repair.

EXAMPLE 4 - SEEDING OF MSCS ONTO ACELLULAR SCAFFOLDS

[0220] In this example, the ability of MSCs to attach, migrate, proliferate, survive, differentiate in to neural, lung epithelial or endothelial cells and produce immunomodulatory factors following placement of these cells onto acellular (AC) lung scaffolds which are produced from human natural lung is examined. With a goal of producing engineered lung tissue, lung scaffold may be used to determine if RA-primed MSCs have the capacity to replicate neuronal processes found in the lung and to determine if the lung scaffold influences differentiation of cells to neuronal, lung epithelium or endothelial cell lineages. AC scaffolds are lungs whose original cells have been destroyed by exposure to detergents and physical methods of removing cells and cell debris (Fox et al, 2005). This creates a lung scaffold from the skeleton of the lungs themselves.

[0221] The development of equipment and procedures to produce whole AC human lung scaffolds has been realized (Arand et al, 1992). Multiphoton microscopy of these AC scaffolds has indicated that the extracellular matrix components in the scaffold are primarily collagen- 1 and elastin and that the natural architecture of the lung is maintained in these scaffolds (Arand et al, 1992).

[0222] Stem cell transplantation is a therapeutic strategy which has the potential to replace damaged cells as well as modify the environment through production of trophic factors. A variety of stem cell sources such as adult human umbilical cord blood, bone marrow or the brain itself have been considered for development of cell-therapy for a variety of diseases. In order to develop MSC-based therapies for lung, brain or other soft tissues there needs to be a consistent cell selection and development of differentiation strategies to produce cells of specific lineages in order to be able to realize their clinical benefits. Practical design for any stem-cell based clinical therapy should include: (1) identification of cell surface markers which allow for consistent isolation of cells at numbers and purity sufficient for use in a therapy, (2) selection of procedures or priming treatments that target differentiation at high efficiency to specific lineages and (3) consideration of the ability of the cells to migrate to damaged areas without the need for direct implantation where the injury has occurred.

[0223] It is known that MSCs stem cells have the capacity for precise migration during embryogenesis and later in life in response to injury, using the CXCR4/SDF-1 (Sawada et al, 2008) pathway. Because of this, subpopulations of peripheral blood mononuclear cells (MNC) were examined for expression of stem cell markers of immaturity combined with expression of CXCR4 and examined neural lineage potential of these subpopulations after RA-priming. A population of CXCR4+ CD133+ ABCG2+ cells were identified that had a high degree of neuronal lineage differentiation efficiency and migrated using a CXCR4/SDF-1 mechanism. Implantation of RA-primed CXCR4+ CD133+ ABCG2+ cells into the lateral ventricle of uninjured or TBI rats resulted in survival of cells, migration to the injury site and differentiation of cells after implantation.

[0224] The ability to obtain these MSCs from an easily accessible source, such as peripheral blood, makes them an excellent candidate for use as an autologous stem cell therapy for the treatment of traumatic brain injury and neurodegenerative disorders. This MSC cell population also has potential for use to treat traumatic brain injury for lung or other soft tissue injuries.

EXAMPLE 5 - ISOLATION OF MSC SUBSETS

[0225] CD105+CD90+CD29+ MSCs and MSC subsets were isolated as previously described from peripheral blood buffy coats (Sawada et al, 2008) or from human bone marrow. The mononuclear (MNC) fraction was isolated using Ficoll density gradient separation medium (Amersham-Biosciences, Piscataway, NJ, USA). Subpopulations of MNC were isolated by counter-current centrifugal elutriation using a Beckmann J6M elutriator (Beckman Instruments) in a Sanderson chamber. A Masterplex peristaltic pump (Cole Parmer Instruments) was used to provide the counter current flow. RPMI 1640 medium supplemented with 2 mM glutamine, 100 U penicillin G, 100 μg/mL streptomycin, and 10% donor-derived autologous serum was used as the elutriation medium. 3-6 x 10⁶ cells were loaded at 3000 RPM, and cell fractions were isolated using a step-wise reduction of rotor speed and medium flow to allow for collection of subpopulations of MNC based on cell size and density.

[0226] For tracking purposes MSCs were labeled with different CFSE colors for cell tracking. CFDA, SE is a fixable-cell- permeant, fluorescein-based tracer for very long- term cell labeling. CellTrace Far Red DDAO-SE is a fixable, far- red— fluorescent tracer for very long-term cell labeling. The succinimidyl ester (SE) reactive group forms a strong covalent attachment to primary amines that occur in proteins and other biomolecules on the inside and outside of cells. Deposition of CFSE labeled MSCs, proliferation and dispersal of cells was determined in CFDA-labeled cell constructs as previously described (Sawada et al, 2008; Arand et al, 1992).

EXAMPLE 6 - ANALYSIS OF CELL PHENOTYPES

[0227] Antibodies for phenotyping were conjugated to fluorescein isothiocyanate (FITC), phycoerytherin (PE) or PerCP were purchased from commercial sources and were used as described by each manufacturer. Corresponding immunoglobulin (IgG) matched isotype control antibodies were used to set baseline values for analysis markers. Staining for ABCG2 (Stem Cell Technologies, Vancouver, BC, CANADA), CD133 (Miltenyi Biotech, Auburn, CA, USA) or CXCR4 (BD Biosciences, San Jose, CA, USA) was done with PE conjugated antibodies. MNCs were stained in PBS (Ca- and Mg-free) supplemented with 5% autologous serum. After the final wash, cells were kept at 4°C in neurobasal media prior to culture or cells were fixed with 2% paraformaldehyde before analysis using a FACSAria instrument (BD Biosciences), with acquisition and analysis using the FACSDiva program (BD Biosciences).

[0228] In a subset of experiments, the Becton Dickenson (BD) human TH1/TH2 flow cyometric cytokine bead array kit was used to measure Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-10 (IL-10), Tumor Necrosis Factor (TNF) and Interferon-γ (IFN-γ) protein levels produced by elutriated MNC pre and post RA- priming as described by the manufacturer. Phagocytic capacity of elutriated cells was evaluated using uptake of fluorescent beads as evaluated by uptake of fluorescent beads. Cell cultures were exposed to 3.5 μg/mL lipopoly saccharide (LPS), or 4 μg/mL phytohemagglutinin-M (PHAM).

EXAMPLE 7 - NEURAL DIFFERENTIATION

[0229] Cells may be cultured in DMEM-LG supplemented with 10% human AB serum, 10-3 M β-mercaptoethanol (β-ΜΕ) (Sigma; St. Louis, MO, USA) + 5 x 10^"7 M all-irans-retinoic acid (RA) (Sigma) for 24 hrs (Sawada et al., 2008). Once cells have been injected into the bioreactor RA primed, 3-6 μιη MSCs may be induced to differentiate using time release Multistage™ (Leonardo Biosystems, Inc., Houston, TX, USA) nanoparticles loaded with 2 mM L-glutamine, B-27 supplement (Invitrogen), 10 μg/mL epidermal growth factor (EGF) and 2.5 μg/mL fibroblast growth factor-beta (FGF-β) and 10% autologous or human AB serum. Viability of cells may be determined using a Molecular Probes LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, San Jose, CA, USA).

[0230] Methods of MSC deposition may be done by infusion of the MSCs into the chambers containing lung scaffold using a catheter method for recellularization by insertion of a set of 2-3 catheters in the main stem bronchus or main bronchi of the acellular lung scaffold. This method has been used previously to place other cell types in the acellular scaffold (Arand et al, 1992). A single aliquot of MSCs may be injected into the acellular lung scaffold and the system may be cultured at 37°C for the duration of the project. When the experiment is to be terminated media and cell culture products may be removed from the bioreactor chambers. The MSC- scaffold construct may be fixed using 2% paraformaldehyde which may be added to the bioreactor chambers. MSC cell supematants and fixed MSC cell constructs are then analyzed for levels of inflammatory mediators produced by MSCs or MSC subsets using ELISA as well as immunostaining for the presence of these factors as has been done previously (Sawada et al, 2008). Initially production of IL-4, IL-10 or IL-13 by MSCs will be examined. Proteomic screens using Mass spectrometry will be performed to determine neural, lung epithelial and lung endothelial phenotypes as previously described (Sawada et al, 2008; Arand et al, 1992). For histological evaluation sections may be stained with hematoxylin and eosin or may be stained with anti-CD 105, CD90 and CD29 (Pharmingen) primary antibodies followed by staining with anti-mouse FITC, PE, or Rhodamine conjugated secondary antibodies (Molecular Probes) as previously described. Cells may be fixed with paraformaldehyde (PAF) before analysis using a FACSAria instrument (BD Biosciences), with acquisition and analysis using the FACSDiva program (BD Biosciences). MSC may be seeded onto the scaffold in the bioreactor and adhered cells may be fixed using PAF.

[0231] Immunohistochemistry: The MSC-lung scaffold construct may be fixed with 2% (wt./vol.) paraformaldehyde for 30 min at 37°C and held at 4°C until assay. Following transfer, the construct may be washed in phosphate buffered saline (PBS) and then frozen sections may be cut. Sections may be permeabilized in 1% BD permeabilizing solution (BD Biosciences) for 10 min with a final wash in Tris-buffered saline (TBS). Nonspecific binding was blocked by a 1-hr treatment in TBS plus 0.1% w/v Tween containing defatted milk powder (30 mg/mL). Cells were incubated for 1 hr at 37°C with one of the following primary antibodies (diluted in blocking buffer): nestin (Chemicon, dilution 1:200), anticholine acetyl transferase (CHAT, Chemicon, 1:250 dilution), galactocerebroside (AGAL, Chemicon, 1 :200 dilution), glial fibrillary acidic protein (GFAP, Dako, Glostrup, Denmark, 1:500 dilution), tyrosine hydroxylase (Cell Signaling Technologies, Danvers, MA, USA and Chemicon, 1:200 dilution), type III tubulin (Tuj l (Eurogentec, Southampton, Hampshire, UNITED KINGDOM, 1 : 1,500 dilution), microtubule associated protein 1 β (MAP-1 β Chemicon, 1:200 dilution) and stage specific embryonic antigen- 1 (SSEA-1, Chemicon, 1:300 dilution) or markers of other cell lineages such as lung epithelium or endothelium as previously described (Arand et al, 1992). [0232] After three washes in TBS, cells may be incubated in secondary antibodies conjugated to fluorescein isothiocyanate (FITC), rhodamine, or Cy5 anti-mouse, anti-rat, or anti-rabbit IgGs (1:500 dilution) for 1 hr at 37°C, then stored at 4°C until analyzed. Use of isotype matched controls and omission of primary antibodies will serve as negative controls and resulted in no detectable staining in confocal analysis or less than 2% background staining for flow cytometry analysis of samples. The preparations will then be mounted in Slow Fade GOLD with DAPI (Molecular Probes) and observed using an LSM 510 Meta advanced laser scanning confocal microscope (Zeiss, Thornwood, NY, USA).

[0233] MSCs may be evaluated for cell adhesion, migration in then scaffold, cytokine production and apoptosis induction using a conventional TUNEL assay. Sections of the fixed MSC-lung scaffold construct may be frozen sectioned and MSCs may be examined for production of immunomodulatory factors. Location and extent of fluorescent labels may be examined using a Nikon T300 Inverted Fluorescent microscope (Nikon Corp., Melville, NY, USA).

[0234] Statistical Analysis: For cell phenotype analysis 10,000 cells were collected for each sample. Statistical analysis may be performed using GraphPad InSTAT software (version 2003). Mean values and standard deviation between data collected for MSCs may be determined. Analysis of variance (ANOVA) may be performed and data subjected to Tukey Kramer multiple comparison test. Mean differences in the values are considered significant when p is less than 0.05.

EXAMPLE 8 - MESOPOROUS SILICON DRUG DELIVERY VECTORS

[0235] MSVs were designed, above all, for delivery of specific drugs during the treatment of cancer (Ferrari, 2005). Their particular structures and material composition allow for the attachment and encapsulation of different type of drugs. The payload can be chemically attached on the particle surface through a previous chemical functionalization or they can be physically loaded in the pores of the particle; this approach offers the opportunity to deliver different types of payloads at the same time but also allows for the sequential delivery of different substances: a particle can be loaded with three or more types of drugs, the first , for instance, can be physically loaded in the pores of the vector, the second one can be encapsulated in liposomes that in turn are loaded in the pores of the particle, and finally a third drug can be chemically attached on the surface. Depending on the biodegradation rate of the MSVs and so on the physical and chemical conditions of the environment the different drugs can be released in different moment, allowing for a sequential and multiple drug release. Focusing on the example just given, it is straightforward to understand that the first physically loaded drug would be the last to be released finding itself in the bottom of the pores covered by the second physically loaded drug; moreover, the release of drug encapsulated in liposomes or micelles will depend of the degradation on the phospholipidic shell that surround it. Of course, the types of drugs that can be loaded on MSVs can be of different nature, such as, chemical or physical; they can release substances able to kill the target cells throughout chemical reactions, enzimatic reactions or physical phenomena like thermal ablation,meaning a treatment that employs special metal nanoparticles that once excited with an opportune electromagnetic source they start increasing their temperature until they reach a level that damages and kills cells, and many other technique that exploit particle activation or photon emission to create an environment harmful for target cells.

[0236] When dealing with cancer and its treatment it becomes of primary importance to be able to reach the zone of interest with the drugs; this is one of the main problems that has been encountered by researcher committed to this difficult challenge. The pharmacokinetics of the drug injected in the body must be studied in details to ensure that the appropriate therapy reaches the tumor in an appropriate amount without being cleared too quickly by lever or kidneys, for example, or without reaching tissue or organs that may be seriously damage by the drug. After intravenous injection MSVs can be sequestered from the blood flow in different ways: they can take and follow smaller and smaller capillaries until they are extravasated from them, they can be swallowed by phagocytic cells but that can also leave the blood vessels thanks to the presence of fenestration especially in the cancer region where they are usually more frequent and finally they can also adhere to blood vessel walls.

[0237] Another interesting strategy for targeting cancer cells consists in the coating of particles with different targeting moieties that allow the attachment of particles to specific target present on cancer cells, keeping the porous structure the drug to annihilate them once attached.

[0238] MSVs can be created in different shape, they can be spherical, cylindrical, hemispherical or discoidal depending on the fabrication process employed. It has been noticed that also the shape of the MSVs is crucial for their uptake by different tissues and organs (Gilbert et al, 2009). The study conducted to address and answer this aspect showed that spherical particles are the ones able to reach the majority of the organs with the lowest loss in the liver.

[0239] Although MSVs were designed initially for cancer treatment it turned out that they could have been used for other applications too. Studies are now conducted to use them in the imaging field (Ferrari, 2005). They can be loaded for example with contrast agents for magnetic resonance imaging (MRI) and other imaging techniques.

[0240] Thinking of new applications for MSVs other than cancer therapy took us to design a way to exploit them in tissue engineering especially for growth factor distribution that represents the originality of this research job as explained below.

[0241] Growth factors are in general substances capable of triggering and guide cell proliferation, differentiation and growth; they are above all proteins but they could be represented by hormone too. Nerve growth factor (NGF), discovered by Nobel Laureate Rita Levi-Montalcini ,was just the first one to be identified, today we can count many of them as, for instance, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) or hepatocyte growth factor and so on. Growth factor are molecules, usually proteins or steroid hormone, able to give the cells different instructions to allow for their proliferation or differentiation, depending on the situation and the needs.

[0242] Tissue engineering deals with techniques and methods to induce regeneration; using cells to repair damages that the body can not normally repair, or recreate biological functions thus to substitute the ones that an organs can no more support are the goals of tissue engineering. The idea of using cells as mentioned above is brilliant and may constitute the solution to this issue, but cells alone are not able to proliferate and differentiate and the required rate and quantity thus, more often, the employment of growth factors is essential. In addition, it must be mentioned,as stated in the previous chapter, that cell differentiation depends also on the environmental conditions that surround them, so not only growth factor presence and type but also ECM composition and morphology; this explains why we need a well-decellularized lung to achieve a good cell differentiation and proliferation.

[0243] Another important fact that is worth to be mentioned and that constitutes one of the major issue dealing with growth factors research is their delivery system. Several studies demonstrated that large doses of growth factors, in solution form, directly to the site where they were required did not give the results expected; growth factors indeed are cleared quite quickly in the body and only a small amount of the injected may reach the interested part. They do not show, in general, long-range diffusion through the extracellular matrix, underlying their tendency to have a local effects and a slow diffusion. Once considered these issue, it is straightforward that to have an efficient distribution of growth factors a good delivery system must be found; here the solutions reported in literature are copious. Up to the present, the best way to deliver these molecules seems to be the employment of biocompatible scaffold materials, such as artificial polymers or natural ones such as gelatin. Growth factor should also be time- release adjustable, that means, to have the possibility to tune the time and the amount per time that these devices need to release the molecules. In addition, for the majority of the studies, more than one growth factor as to be employed requiring the possibility of simultaneous or sequential, depending on the situation, delivery of multiple growth factors.

[0244] In literature several solution have been presented, biocompatible scaffolds is only one of the many existing: there are techniques that exploit the change in Ph or temperature to release growth factor, others that, thanks to a greater concentration of particular proteinase, in the site of interest, that cleaves the chemical bond keeping the growth factor linked to the delivery system, releases the signaling molecules just where needed. There are also different way of loading the proteins onto the delivery systems; it can be done throughout the exploitation of physical principles or chemical ones depending on the situation where they have to be used. This last observation is of great importance since it has been proven that the method f loading can denature the protein and make the growth factor complete useless for the purpose it was employed for.

[0245] Growth factor are then quite expensive even for a research lab, and this is the reason why for these preliminary studies it was decided to adopt a protein which could have taken their place; was selected as the phantom of the growth factor. This protein was chosen because it is less expensive and having a quite high molecular weight it could have simulate better the worst case of a huge growth factor, given also the fact that it is still unclear which combination and which types of growth factors should be employed to reach the desired recellularization of the acellular lung previously obtained.

[0246] The main idea underlying the delivery system designed for our purpose is the loading of the proper protein in the mesoporous silicon particles (MSVs) (FIG. 18A and FIG. 18B). It is required that the particles are perfused into the lung so that they can spread and can reach the inmost zones. To be sure that those particles can reach even the most difficult place inside the lung a brief study was conducted: MSVs particle were loaded with Rhodamine dye and perfused into an acellular lung, then images with an IVIS imaging system were taken and compared to the control images taken before the particle perfusion; then discoidal and spherical particles loaded with Rhodamine were used to see if effectively the discoidal ones could reach and accumulate better in the lung than the spherical ones. To evaluated the amount of particles in the tissues, Inductive Coupled Plasma (ICP) mass spectroscopy was used in order to evaluate the amount of silicon present in the tissue and estimate the number of particles stacked there. Finally scanning electron microscopy (SEM) micrographs of the tissue coming from lungs perfused with particles were studied to detect the presence of internal MSVs (FIG. 21A, FIG. 21B, and FIG. 21C).

[0247] Once proven this capability of the MSVs, the research moved and focused on the method of loading and releasing of the desired protein. There are basically two ways by which these particles could be loaded with molecules, in this case growth factors; as stated above molecules can physically penetrate the porous silicon particle and remain there for the time required for the release, at least this is what it is expected and desired, or they can be chemically attach to the particle, and in this case the release can be accomplished by means of other strategies like the employment of special molecules such as specific enzymes able to cleave the binding that keeps the growth factor attached to the particle. For this particular work the physical approach was preferred to the chemical one.

[0248] Particles used for these experiments were all discoidal; they were initially suspended in Isopropanol were disposed in small tubes and the tubes were eased down in a vacuum chamber to let the isopropanol evaporate; once dried, the particles were then suspended in different concentration of albumin, than sonicated to mix and help the particles to spread in the solution containing the protein and finally laid on a plate shaker for half an hour in order to help the loading of the protein into the MSVs.

[0249] To check the loading and obtain a curve of the mass of albumin into the MSVs versus the mass used for the loading, the particles suspended in the albumin loading solution were spun down and the supernatant was studied with a platereader that allows to perform a fluorescence analysis and thus evaluate the amount of protein not loaded. Once discovered the amount of protein not loaded it was easy to find the percentage that instead was kept by the MSVs. Than, thanks to the different concentrations of albumin tested, a loading curve was obtained that showed the loaded mass versus the initial one used for each case. To study the release the cases for which the maximum loading was reached were kept and used as reference points: the amount of particles and albumin were indeed increase since, for the release, higher volumes and quantities are required. The quantities were increased following a linear behavior, meaning that if the maximum load was obtained with 3J-Lg of albumin for 5 million MSVs, the released was then studied using 15J-Lg of albumin for 25 million MSVs using the ratios: 3J-Lg+5 x 106 l\18Vs = 15J-Lg+25 x 106 MSVs (FIG. 22).

[0250] Once the MSVs were loaded they were immersed in a solution made of 10% of Fetal Bovine Serum (FBS) in IX PBS that has a pH of about 7, simulating the basic human body physiological condition. The supernatant concentration of albumin was then kept measured with time for about 10 days. As said above, knowing the concentration of the supernatant, the volume of the solution and the mass loaded on the particles allows for the evaluation of the mass released of each sample collected during the experiment. It is worth to highlight that the release experiment just mentioned was conducted just to understand and verify the possibility of loading and release of big proteins such as albumin or growth factors with MSVs; it is obvious how the real release conditions could be totally different while perfusing the albumin loaded MSVs into the lung during recellularization: the presence of a solid acellular scaffold and of few cells seeded for the recellularization among with the higher volumes of solutions and a higher number of particles contribute to change the environment where these experiments were performed during this work.

[0251] To study the behavior of the cells of lungs in vitro with and without growth factors 2 assay kits were used: the Fibrin Gel In Vitro Angiogenesis Assay Kit (a product of EMD Millipore Corporation) and the Colorimetric (MTT) Kit for Cell Survival and Proliferation (EMD Millipore Corporation). These kits were employed with two types of human cells: the Human Lung Microvascular Endothelial Cells (HLMVEC) (purchased from Cell Applications, Inc.) and the Human Pulmonary Alveolar Epithelial Cells (HPAEpiC) (Science Cell™ Research Laboratories).

[0252] Human lung microvascular endothelial cells are primary endothelial cells isolated from normal adult human lung capillaries; also human pulmonary alveolar epithelial cells, containing alveolar type I (AEC I) and alveolar type II (AEC II), responsible for lining more than 99% of the internal surface of the lung, were isolated from human lung tissue. The kits were purchased with the intention to study cell behaviors in the presence of certain growth factors.

[0253] The kits used for these studies facilitated two separate goals: the fibrin gel in vitro angiogenesis assay kit was chosen to demonstrate whether cells were able to migrate and start to differentiate and thus showing, as the name suggests, tube and capillary formation; it exploits a fibrin gel since endothelial cells can rapidly align and make interconnections and finally display tube formation, a process that involves cell adhesion, differentiation, migration and proliferation; the colorimetric (MTT) kit for cell survival and proliferation instead is designed to study the proliferation of a cell culture under specific conditions; MTT is a yellow substrate that yields formazan product which has a dark blue color; this is due to the ability of only living cells to cleave MTT, explaining its purpose of studying the proliferation of a cell culture.

[0254] For these tests three types of growth factor were adopted: the Recombinant Human Fibroblast Growth Factor-basic (FGFb, Life Technologies), the Human Recombinant Derived Growth Factor (PDGF, Life Technologies) and finally the Human Vascular Endothelial Growth Factor (VEGF, sold by Sigma-Aldrich). Both three growth factors play an important role for cell proliferation and differentiation, especially PDGF and VEGF are fundamental in vessel formation, that is the reason why it was decided to use a kit aimed to study agiogenesis using human lung epithelial cells. The results obtained form these studies may provide significant clues on the way the lung should be recellularized using MSVs as growth factor delivery system. Knowing how a certain growth factor acts on cells of the lung and which kind of cells have the fundamental cell behavior in the lung represents now a major filed of research for biologists. AEC II cells, for instance, may have an essential role in a future recellularization since they are believed to have self renewal ability and they seem to be able to reenter the cell cycle and differentiate in other type of cells such as AEC I.

[0255] Cells were cultured in plate wells following the manufacturer's protocols in triplicate for each experiment. Once set up, different concentration of various growth factors were administered to the cell cultures. Each experiment was constituted by a single cell type (i.e. either HLMVEC or HPAEpiC) and a particular growth factor at a specific concentration.

[0256] As stated above, each experiment was performed in triplicate, including the controls, which did not receive growth factors. For each growth factor type, three different doses (or concentrations) were administrated to each type of cell in triplicate. That means that 48 wells were examined: having two different type of cells (HL1VIVEC and HPAEpiC), and, for each, a different concentration of a growth factor type administrated, more exactly three per growth factor type, plus the controls, everything done in triplicate, gives 24; times 2, since two different assay kits were used, gives 48. [0257] The first study conducted for the growth factor distribution project was the perfusion of solution containing particles in the acellular lung scaffold (FIG. 19A, FIG. 19B, and FIG. 19C). The IviSVs used for this study were discoidal since from preliminary results they ended up to be more dispersed in tissues than the others and they were suspended in a IX PBS solution. To see where the particles, loaded with rhodamine spread after their perfusion it was employed the IVIS imaging system mentioned above: a normal pig lung was used as control and a normal and a decellularized lung as the main samples. It was interesting to observe how the results were markedly different: the control lung appeared to be completely full of particle, while the acellular one was almost empty.

[0258] Since IVIS images gave unexpected results, to verify whether a significant amount of particles spread in the acellular organ, ICP mass spectroscopy was employed. In this case discoidal and spherical particles were perfused in different acellular pig lungs in solution of Hespan® and blood (FIG. 20).

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[0304] YI, T and SONG, SU, "Immunomodulatory properties of mesenchymal stem cells and their therapeutic applications," Arch. Pharm. Res., 35(2):213-221 (Feb. 2012). [0305] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. All references (including publications, patent applications and patents) cited herein are incorporated herein by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0306] The description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing," with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of," or "substantially comprises," that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0307] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the invention have been described herein in terms of illustrative embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods, and/or the steps or the sequence of steps of the methods without departing from the spirit, scope, and concept of the invention. More specifically, it will be apparent that certain compounds, which are chemically- and/or physiologically-related, may be substituted for one or more of the compounds described herein, while still achieving the same or similar results. All such substitutions and/or modifications, as apparent to one or more of ordinary skill in the relevant arts, are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

CLAIMS:

1. A method for producing a decellularized organ scaffold comprising:

1) aerating an isolated organ in a selected medium to disrupt cell membranes without destroying the interstitial structure of the organ;

2) treating the isolated organ in an alkaline solution having a detergent at a concentration effective to extract cellular material without dissolving the interstitial structure of the organ; and

3) washing the isolated organ in distilled water to remove cellular debris without removing the interstitial structure of the organ until the organ is substantially free of the cellular material, to thereby produce a decellularized organ scaffold.

2. The method of claim 1 , wherein the organ is a mammalian lung.

3. The method of claim 1 or 2, wherein the organ is a human lung.

4. The method of any preceding claim, further comprising equilibrating the decellularized organ scaffold in a pharmaceutically-acceptable buffer solution.

5. The method of any preceding claim, wherein the rate of aeration is controllable in a time- or concentration-dependent manner.

6. The method of any preceding claim, wherein the rate of aeration is sufficient to form detergent micelles in the alkaline solution.

7. The method of any preceding claim, wherein the rate of aeration is sufficient to bubble off the detergent-containing alkaline solution from the residual decellularlized organ.

8. The method of any preceding claim, wherein the alkaline solution comprises a detergent selected from the group consisting of sodium dodecyl sulfate (SDS), Triton X-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, and Triton DF-16, monolaurate (Tween 20), monopalmitate (Tween 40), monooleate

(Tween 80), polyoxyethylene-23-lauryl ether (Brij 35), polyoxyethylene ether W- 1 (Polyox), sodium cholate, deoxycholates, CHAPS, saponin, n-Decyl β-D- glucopuranoside, n-heptyl β-D glucopyranoside, n-Octyl a-D-glucopyranoside and Nonidet P-40.

9. The method of any preceding claim, wherein the detergent comprises SDS.

10. The method of any preceding claim, wherein the step of washing comprises rotating the isolated organ kidney in distilled water in a stirring vessel.

11. An acellular mammalian organ prepared by the method of claim 1.

12. The acellular mammalian organ of claim 11, adapted and configured as part of a therapeutic kit that comprises the composition, and at least a first set of instructions for preparing the organ for human transplantation.

13. A composition comprising:

(a) a population of mesoporous silicon (pSi) nano- or micro-particles; and

(b) the acellular mammalian organ of claim 11.

14. The composition of claim 14, further comprising at least a first therapeutic agent comprised within the population of pSi nano- or micro-particles.

15. The composition in accordance with claim 13 or 14, further comprising an anti- rejection compound, an anti-inflammatory compound, or a combination thereof.

16. Use of a composition in accordance with any one of claims 13 to 15, in the manufacture of a medicament for treating or ameliorating at least one symptom of organ failure in a mammalian subject.

17. Use in accordance with claim 16, wherein the mammalian subject is a human, a non-human primate, a companion animal, an exotic, or a livestock.