Controlled release implants for biologics and corresponding therapeutic approaches

Abstract

An implant, such as a pharma- ceutically acceptable implant, comprising a xerogel, a biologic, and at least one dehydration stabilizer.

Description

[Technical field]

[0001] The present invention relates to an implant for a biologic. In particular, the present invention relates to a pharma- ceutically acceptable implant for the controlled release of a biologic, such as a viral vector. The present invention also relates to corresponding methods of treatment and uses. [Background technology]

[0002] Controlled delivery of therapeutic agents is an extensive area of ​​research, particularly in recent years, for biologics, which offers the potential for improved treatment, easier administration, increased compliance, fewer side effects, and improved outcomes.

[0003] However, delivery of ocular therapeutics to the eye remains a challenge, and the effectiveness of these therapies is hindered by a variety of factors, most of which relate to the eye being an immunologically privileged organ and its limited size.

[0004] Although therapeutically effective doses may be required to treat eye diseases, it may not be feasible to administer large amounts of a therapeutic agent to the eye without causing inflammation, or to administer the therapeutic agent more frequently.

[0005] For example, gene therapy is a fairly new but promising treatment modality that is being explored for the treatment of eye diseases. The basic concept of gene therapy is to solve the genetic problem at its source. For example, if a mutation in a gene results in the production of a malfunctioning protein, which then causes a disease, gene therapy can be used to deliver a copy of this gene that does not contain the harmful mutation, thereby producing a functional protein. Not only can the genetic problem be addressed, but any kind of therapeutic protein can also be produced in the patient via a vector to treat a particular disease.

[0006] In such treatments, viral vectors encoding a specific therapeutic agent are most often administered. These viral vectors have the ability to replicate and cause the expression of a therapeutic protein. Very often, the expression of the therapeutic protein contained in the viral vector or the viral vector itself induces inflammation, which reduces the expression of the therapeutic protein contained in the viral vector and, as a result, reduces the efficacy of the gene therapy. Thus, these approaches suffer from a major bottleneck in that inflammation cannot be avoided when delivering gene therapy vectors. Several methods have been studied to solve this problem, including the use of steroids, which are known to be anti-inflammatory agents. However, administering steroids to the eye can increase intraocular pressure, which can significantly increase the risk of developing intraocular pressure-related eye diseases such as glaucoma.

[0007] Complex biologics, such as viral vectors, are difficult to stabilize in more complex formulations. Temperature, organic solvents, other chemicals, and removal of water pose significant challenges to the formulation of biologics, especially complex biologics. Summary of the Invention

[0008] It is an object and one aspect of the present invention to stabilize a biologic, such as a viral vector, in a pharma-ceutically acceptable implant.

[0009] It is an object and one aspect of the present invention to provide a pharma- ceutically acceptable implant that contains a biologic.

[0010] It is an object and one aspect of the present invention to stabilize biologics, such as viral vectors, in pharma- ceutically acceptable implants having polymer networks.

[0011] It is an object and one aspect of the present invention to stabilize a biologic, such as a viral vector, during the manufacture of an implant, such as an implant having a polymer network.

[0012] It is an object and an aspect of the present invention to provide a pharma- ceutically acceptable implant that contains a biologic within a polymer network, allowing for the controlled release of the biologic, such as a viral vector, from the pharma- ceutically acceptable implant.

[0013] It is an object and one aspect of the present invention to provide a pharma- ceutically acceptable implant comprising a viral vector for ocular gene therapy.

[0014] It is an object and one aspect of the present invention to deliver biologics, such as viral vectors, to the eye while controlling inflammation.

[0015] It is an object and one aspect of the present invention to deliver viral vectors to the eye for expression of therapeutic proteins while controlling inflammation.

[0016] It is an object and one aspect of the present invention to deliver viral vectors to the eye for expression of therapeutic proteins while controlling the immune response.

[0017] It is an object and one aspect of the present invention to deliver viral vectors to the eye for expression of therapeutic proteins while controlling the adaptive immune response, such as the humoral immune response. It is an object and one aspect of the present invention to treat inflammation of the eye, particularly inflammation due to the adaptive immune response.

[0018] Some embodiments of the present disclosure are directed to an implant, such as a pharma- ceutically acceptable implant, comprising a xerogel, a biologic, and at least one dehydration stabilizer.

[0019] Some embodiments of the present disclosure are directed, in part, to a pharma- ceutically acceptable implant for the controlled release of a biologic, wherein the controlled release is characterized by the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 2 days.

[0020] Some aspects of the present disclosure are directed to methods of treating an eye disease, such as a genetic eye disease, comprising administering to a subject a pharma- ceutically acceptable implant of the present invention or a corresponding use to a subject.

[0021] Some aspects of the present disclosure are directed to methods of controlling inflammation in treating ocular diseases, such as genetic ocular diseases, comprising administering to a subject a pharma- ceutically acceptable implant of the present invention or a corresponding use to a subject.

[0022] Some aspects of the present disclosure are directed to methods of controlling immune responses, such as adaptive immune responses, in treating ocular diseases, such as genetic ocular diseases, comprising administering to a subject a pharma- ceutically acceptable implant of the present invention, or a corresponding use to a subject.Some aspects of the present disclosure are directed to providing an effective method in treating ocular diseases, such as genetic ocular diseases, comprising administering to a subject a pharma- ceutically acceptable implant of the present invention, or a corresponding use to a subject.

[0023] Some embodiments of the present disclosure are directed to the use of at least one dehydration stabilizer to protect the biologic from damage during processes in which the biologic is directly exposed to organic solvents.

[0024] Some embodiments of the present disclosure are directed to methods of protecting a biologic from damage during a process in which the biologic is directly exposed to an organic solvent, the method comprising mixing the biologic with at least one dehydration stabilizer prior to directly exposing the biologic to an organic solvent.

[0025] Some aspects of the present disclosure are directed to methods of making a pharma- ceutically acceptable implant comprising a biologic comprising: (A) forming an organogel comprising the biologic, comprising forming a matrix comprising at least two covalently crosslinked multi-arm precursors in an organic solvent in the presence of the biologic; and (B) forming a xerogel, comprising removing the organic solvent.

[0026] The present invention also provides a method of preparing a pharma- ceutically acceptable implant for the controlled release of a total amount of a biologic, comprising the steps of: (a) selecting the total (w / w) % of carbohydrates, sugar alcohols, or a combination thereof; (b) selecting the molecular weight between crosslinks in the xerogel; (c) selecting a percentage (w / w) of the total particles that comprises a mixture of the biologic and a carbohydrate, sugar alcohol, or combination thereof; (d) selecting a (w / w) % of the total number of multi-arm precursors; (e) selecting the ratio of (c) to (d); and / or (f) selecting a molar ratio of (fi) to (f-ii), (fi) a first reactive group contained in a second multi-arm precursor; and (f-ii) selecting a molar ratio of the second reactive group contained in the third multi-arm precursor; Methods are directed to where the (w / w)% is based on the weight of the pharma- ceutically acceptable implant.

[0027] The present invention also provides a method of preparing a pharma- ceutically acceptable implant for the controlled release of a total amount of a biologic, comprising the steps of: (a) selecting the total (w / w) % of carbohydrates, sugar alcohols, or a combination thereof; (b) selecting the molecular weight between crosslinks in the xerogel; (c) selecting a percentage (w / w) of the total particles that comprises a mixture of the biologic and a carbohydrate, sugar alcohol, or combination thereof; (d) selecting a (w / w) % of the total number of multi-arm precursors; (e) selecting the ratio of (c) to (d); (f) Domestic violence 90 D such as particle size 90 Selecting a particle size, the particles comprising a mixture of the biologic and at least one dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof; and / or (g) selecting a molar ratio of (gi) to (g-ii), (gi) a first reactive group contained in a second multi-arm precursor; and (g-ii) a second reactive group included in a third multi-arm precursor; and selecting a molar ratio of Methods are directed to where the (w / w)% is based on the weight of the pharma- ceutically acceptable implant.

[0028] Some aspects of the present disclosure are directed to methods of treating ocular inflammation comprising administering a therapeutically effective amount of a tyrosine kinase inhibitor to the eye of a subject in need thereof.

[0029] definition As used herein, a "precursor" refers to a polymer that meets all of the following requirements: (i) it is soluble in an organic solvent; (ii) it is capable of reacting with other precursors; and (iii) it is non-reactive with biological agents.

[0030] The term "polymer network" describes a structure formed from at least two precursors (of the same or different molecular structure and of the same or different molecular weight) crosslinked to each other. Types of precursors suitable for the purposes of the present invention are disclosed herein. The term "polymer network" is used interchangeably with the term "matrix".

[0031] As used herein, "organogel" refers to a three-dimensional polymer network of at least two precursors that are covalently crosslinked together in the presence of an organic solvent and still contain the organic solvent (in wet form). As used herein, an "organic solvent" is a carbon-based material that is liquid at room temperature and pressure. Such organic solvents can include methylene chloride, dimethyl carbonate, acetone, acetonitrile, ethyl acetate, and tetrahydrofuran.

[0032] "Xerogel" in the simplest sense refers to a dry organogel. A "xerogel" is at least two multi-arm precursors that are covalently crosslinked with each other to form a polymer network, in a dry state. Thus, in certain embodiments, a xerogel in the context of the present invention may contain less than about 5% by weight, 4% by weight, 3% by weight, 2% by weight or 1% by weight of water, such as less than 2% by weight, such as less than 1% by weight of water. The water content of an implant in a dry / dried state can be measured, for example, by Karl Fischer coulometry. Thus, in certain embodiments, a xerogel in the context of the present invention may contain less than 2% by weight of organic solvent, such as less than 1% by weight of organic solvent.

[0033] As used herein, the term "hydrogel" refers to a hydrated xerogel. When placed under physiological conditions, such as pH 7.2-7.4 and 37°C, the xerogel becomes hydrated and is called a hydrogel as described above. Due to its high water content, the hydrogel is soft and flexible, and thus closely resembles natural tissue. In the present invention, the term "hydrogel" is used to refer to a xerogel in a hydrated state (e.g., under physiological conditions) that contains water.

[0034] The term "implant" as used herein refers to a xerogel having any given shape (as disclosed herein). Thus, an implant is a body that includes a xerogel and has entrapped therein an active agent, specifically any biologic (as disclosed herein) in the form of a mixture with at least one dehydration stabilizer.

[0035] An "in situ implant" according to the present invention refers to an implant formed in vivo from a hydrogel precursor composition, including a biologic, that forms a hydrogel upon injection in vivo.

[0036] The term "pharmaceutically acceptable implant" refers to an implant that can be administered to a subject. When administered to a human or animal body, for example, to the vitreous humor of the eye (also called the "vitreous cavity" or "vitreous body"), the "pharmaceutically acceptable implant" remains there for a period of time during which it releases an active agent into the surrounding environment. The implant may have any predefined shape (as disclosed herein) before injection, which shape is maintained to some extent once the implant is placed at the desired location, but as further disclosed herein, the dimensions of the implant (e.g., length and / or diameter) may change after administration due to hydration. In other words, it is not a solution or suspension that is injected into the eye, but a preformed coherent body. Thus, the "pharmaceutically acceptable implant" is fully formed as disclosed herein before administration, and is not created in situ at the desired location in the eye (as can also be done with suitable formulations in general) in embodiments of the present invention. The term "implant" or "pharmaceutical acceptable implant" is used herein to refer to an implant comprising a xerogel and thus in a dried and / or dehydrated state, i.e., after the implant has been manufactured and dried and immediately prior to being loaded into a needle or after being loaded into a needle as disclosed herein, or to an implant manufactured in a dry state without the need for dehydration. When a pharmaceutical acceptable implant is administered to the eye or otherwise immersed in an aqueous environment (e.g., in vitro), the implant becomes hydrated under physiological conditions and is thereafter used to refer to an implant comprising a hydrogel or a pharmaceutical acceptable implant. When dimensions (i.e., length, diameter, or volume) of an implant or pharmaceutical acceptable implant in a hydrated state are reported herein, these dimensions are measured at various specified time points after the implant or pharmaceutical acceptable implant is immersed in an aqueous solution under physiological conditions, such as pH 7.2-7.4 at 37°C.Whenever dimensions of an implant or pharma- ceutically acceptable implant are reported herein in a dry state, these dimensions are measured after the implant is completely dry (thus, in certain embodiments, the implant or pharma-ceutically acceptable implant contains no more than about 5%, 4%, 3%, 2%, or 1% water or organic solvent by weight) and ready to be loaded into a needle for subsequent administration.

[0037] In certain embodiments of the present invention, the term "fiber" characterizes an object having a generally elongated shape, in which the implant or pharma- ceutical acceptable implant is formed. Specific dimensions of the implants of the present invention are disclosed herein. The fiber may have a cylindrical or essentially cylindrical shape. The cross-sectional area of ​​the fiber or implant may be either circular or essentially circular, but in certain embodiments may be elliptical or oval, or in other embodiments may be of different geometric shapes, such as a cross, a star, or other shapes disclosed herein.

[0038] The term "biodegradable" refers to a material or object (such as an ocular implant according to the present invention) that degrades in vivo, i.e. when placed in the human or animal body, or in vitro, when immersed in an aqueous solution under physiological conditions, such as pH 7.2-7.4 at 37°C. In the context of the present invention, as disclosed in detail herein below, the implant slowly biodegrades over time when deposited in the eye, e.g., in the vitreous humor. In certain embodiments, biodegradation is at least partially achieved by ester hydrolysis in the aqueous environment of the vitreous. The implant slowly dissolves until it is completely resorbed and no longer visible in the vitreous.

[0039] As used herein, the term "adeno-associated virus" or "AAV" includes, but is not limited to, all serotypes, such as AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, from any species. For further details on AAV serotypes, phylogenetic groups, and any other AAVs, see, for example, Gao et al. (J. Virol. 78:6381 (2004), Morris et al. (Virol. 33:375 (2004), and FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven). Publishers). For example, the AAV may be an AAV derived from a naturally occurring "wild type" virus, an AAV derived from a recombinant AAV (rAAV) genome packaged in a capsid derived from a capsid protein encoded by a naturally occurring capsid gene, and / or an AAV derived from a rAAV genome packaged in a capsid derived from a capsid protein encoded by a non-native capsid cap gene. As used herein, "AAV" can be used to refer to the virus itself or its derivatives. The term encompasses all subtypes and both naturally occurring and recombinant forms unless otherwise specified. In some aspects, the AAV is a non-replicating AAV, e.g., a non-infectious AAV. In some embodiments, the AAV comprises a viral vector.

[0040] AAV vectors can contain additional elements that function in cis or trans. In certain embodiments, the AAV vector containing the vector genome also has expression control elements (e.g., promoters or enhancers) that drive transcription of the donor sequence, such as one or more ITR sequences adjacent to the 5' or 3' end of the donor sequence, constitutive or regulatable control elements, or tissue-specific expression control elements, intron sequences, stuffer or filler polynucleotide sequences, and / or polyadenine sequences located 3' of the donor sequence.

[0041] The terms "polypeptide," "peptide," "protein," and "protein fragment" are used interchangeably herein to refer to at least two amino acids or amino acid analogs covalently linked by a peptide bond or a peptide bond analog. These terms thus include any protein having primary, secondary, tertiary, or quaternary structure, fragments thereof, and fusions thereof.

[0042] The term "release" (and corresponding terms such as "released" and "releasing") as used herein refers to the provision of an agent such as a biologic from an implant of the present invention to a surrounding environment. The surrounding environment may be an in vitro environment or an in vivo environment as described herein. In certain embodiments, the surrounding environment is an ocular tissue such as the vitreous humor and / or the retina and choroid. Thus, whenever an implant or "a pharmaceutically acceptable implant" is described herein as "releasing" or "allowing for (controlled) release" of a biologic such as an AAV or a recombinant protein, this not only means that said biologic is provided directly from the implant while the hydrogel is not yet (fully) biodegraded, but also that said biologic continues to be provided continuously to the surrounding environment after the hydrogel has fully degraded, when the remaining biologic remains in this surrounding environment for an extended period of time and continues to exert its therapeutic effect.

[0043] The term "controlled release" refers to the release of an active agent, specifically a biologic such as an AAV or recombinant protein, from an implant or pharma- ceutically acceptable implant in a predetermined manner, as opposed to immediate release, such as with a bolus injection. Controlled release refers to the amount of biologic released on day 1, the amount released per day thereafter, and the total number of days required for 100% release of the biologic in an aqueous solution under physiological conditions, such as pH 7.2-7.4 and 37°C. Thus, "controlled release" measured under these conditions is considered to be the same as when a pharma-ceutically acceptable implant is administered to a subject in vivo.

[0044] The term "100% release of biologic" should be interpreted as 95% to 100%. This controlled release is achieved by several parameters that are properties of the pharma- ceutically acceptable implant, as disclosed herein. Each such property of the pharma- ceutically acceptable implant can be involved in the controlled release, either alone or in combination with each other.

[0045] As used herein, the term "total amount of biological agent" refers to the total amount of biological agent that is comprised and / or included in the pharmaceutically acceptable implant. Those skilled in the art can assess the total amount of biological agent before including the biological agent in the manufacturing method of the pharmaceutically acceptable implant of the present invention. For example, if the biological agent is a virus, those skilled in the art may assess the total amount of virus using polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA).

[0046] As used herein, the term "heterologous" or "exogenous" refers to a molecule that is not normally found in a given context, e.g., a cell or polypeptide. For example, an exogenous or heterologous molecule may be introduced into a cell and be present only after manipulation of the cell, e.g., by transfection or other genetic engineering, or a heterologous amino acid sequence may be present within a protein that is not found in nature.

[0047] "Zero order" or "substantially zero order" or "near zero order" release is defined as exhibiting a relatively straight line in a graphical representation of the percentage of biologic released versus time. In certain embodiments of the invention, substantially zero order release is defined as the amount of biologic released that is proportional to within 20% of the time elapsed.

[0048] As used herein, a "dehydration stabilizer" is an excipient and / or additive that protects and stabilizes a biologic or non-biologic (including biologic) in dry form or in the absence of water. For example, when the biologic is a protein, the dehydration stabilizer prevents denaturation or aggregation by preserving the tertiary or quaternary structure of said protein.

[0049] The term "ocular" as used in the present invention refers generally to the eye, or to any part or site of the eye (as an "ocular implant" according to the present invention can in principle be administered to any part or site of the eye), or to any disease of the eye (as in one aspect the present invention generally refers to the treatment of any disease of the eye ("ocular disease") of various origins and nature). The present invention, in certain embodiments, is directed to intravitreal injection of an ocular implant (and thus in this case the "ocular implant" is an "intravitreal implant").

[0050] "Controlling inflammation" according to the present invention means limiting inflammation to an acceptable level so that treatment can continue.

[0051] The term "patient" as used herein includes both human and animal patients. Thus, the pharma- ceutically acceptable implants according to the invention are suitable for human or veterinary medical use. In general, a "subject" is an individual (human or animal) to whom an implant according to the invention is administered. A "patient" is a subject in need of treatment for a particular physiological or pathological condition. A "patient" has not necessarily been diagnosed with a particular physiological or pathological condition prior to receiving the implant.

[0052] The molecular weight of the polymer precursors used for the purposes of the present invention and disclosed herein can be determined by analytical methods known in the art. The molecular weight of polyethylene glycol can be determined by any method known in the art, including, for example, gel electrophoresis such as SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), gel permeation chromatography (GPC) including dynamic light scattering (DLS), liquid chromatography (LC), and mass spectrometry such as matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) spectroscopy or electrospray ionization (ESI) mass spectrometry. The molecular weight of the polymers, including the polyethylene glycol precursors disclosed herein, is an average molecular weight (based on the molecular weight distribution of the polymer) and therefore may be indicated by various average values, including weight average molecular weight (Mw) and number average molecular weight (Mn). For the polyethylene glycol precursors used in the present invention, the molecular weight indicated herein is the number average molecular weight (Mn).

[0053] As used herein, the term "day 1" refers to the time point immediately following "day 0." Thus, whenever "day 1" is used, it refers to a day or period of about 24 hours that has already passed.

[0054] As used herein, "anti-drug antibody (ADA) titer" is expressed as a reciprocal dilution. As used herein, ADA encompasses any ADA known to those of skill in the art, including ADAs that are neutralizing antibodies (Nab).

[0055] As used herein, "D 90 "Particle size" refers to a numerical value representing the diameter of a particle, indicating that 90% of the particle distribution contained in the implants of the present invention has a diameter less than said numerical value.

[0056] As used herein, "Dn 90 "Particle size" refers to a numerical value representing the diameter of a particle, indicating that 90% of the particle population distribution contained in an implant of the present invention has a diameter less than said numerical value.

[0057] As used herein, "Dv 90 "Particle size" refers to a numerical value representing the diameter of a particle, indicating that 90% of the particle volume distribution contained in the implants of the present invention has a diameter less than said numerical value.

[0058] As used herein, the term "about" in connection with a measured quantity refers to normal variation in that measured quantity that would be expected by one of ordinary skill in the art in making the measurement and exercising a level of care depending on the purpose of the measurement and the precision of the measuring device.

[0059] The term "at least about" in relation to a measured amount refers to normal variation in the measured amount and an amount greater than the measured amount that would be expected by one of ordinary skill in the art in making the measurement and exercising a level of care depending on the purpose of the measurement and the precision of the measuring device.

[0060] The term "average" as used herein refers to the central or representative value within a set of data (points) and is calculated by dividing the sum of the data (points) by the number of data in the set (i.e., the average value of the data set).

[0061] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0062] The term "and / or" as used herein in expressions such as "A and / or B" is intended to include both "A and B" as well as "A or B."

[0063] As used herein, open terms such as "include," "including," "contain," "containing," and the like, mean "comprising" and are intended to refer to an open-ended list or enumeration of elements, method steps, and the like, and thus are not intended to be limited to the listed elements, method steps, etc., but are intended to include additional, unlisted elements, method steps, etc.

[0064] When used herein in conjunction with a particular value or numerical value, the term "up to" is meant to include the respective value or numerical value.

[0065] As used herein, the terms "from A to B," "from A to B," and "of A to B" are used interchangeably and all refer to a range from A to B, with an upper limit A and a lower limit B.

[0066] Throughout this disclosure, various aspects of the invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed not only each numerical value within the range, but also all possible subranges. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as each numerical value within the range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. A stated range of numerical values ​​is inclusive of the numerical values ​​defining the range and includes each integer within the stated range.

[0067] As used herein, the abbreviation "PBS" means phosphate buffered saline.

[0068] As used herein, the abbreviation "PEG" means polyethylene glycol. [Brief description of the drawings]

[0069] [Figure 1] Analysis of the effect of AAV2 formulations on (A) % transduction efficiency and (B) MFI of GFP+ cells at an MOI of 1x105. [Diagram 2] Analysis of the effect of AAV2 formulations on (A) % transduction efficiency and (B) MFI of GFP+ cells at an MOI of 1x105. [Diagram 3] Subgroup analysis of the effect of AAV2 formulations on (A) % transduction efficiency and (B) MFI of GFP+ cells at an MOI of 1x105. [Figure 4] Effect of formulation on AAV8 transduction efficiency in HEK293T cells at MOI=1.00E+06 (GFP expression rate by FACS) [Diagram 5] Effect of formulation on AAV8 transduction efficiency in HEK293T cells at MOI=1.00E+06 (MFI analysis by FACS) [Figure 6] Effect of formulation on AAV2.7m8 transduction efficiency in HEK293T cells at MOI=5.00E+05 (GFP expression rate by FACS) [Figure 7] Effect of formulation on AAV2.7m8 transduction efficiency in HEK293T cells at MOI=5.00E+05 (MFI analysis by FACS) [Figure 8] Fiber Manufacturing Process A [Figure 9] Fiber Manufacturing Process B [Figure 10A] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1A and 3.1B [Figure 10B] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1C and 3.1D [Figure 10C] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1E and 3.1F [Figure 10D] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1G and 3.1H [Figure 11A]The release profile can be controlled by selecting the SS:SG ratio - groups 3.1I and 3.1J [Figure 11B] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1K, 3.1L, and 3.1M [Figure 11C] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1N, 3.1O, and 3.1P [Figure 11D] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1Q, 3.1R, and 3.1S [Figure 12] The SS:SG ratio can be selected to control the emission profile - groups 3.1T, 3.1U, 3.1V, and 3.1W. [Figure 13] The SS:SG ratio can be selected to control the release profile - groups 3.1X and 3.1Y. [Figure 14] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1Z and 3.1AA [Figure 15] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1AB and 3.1AC [Figure 16] The release profile can be controlled by selecting the SS:SG ratio - groups 3.1AD, 3.1AE and 3.1AF [Figure 17] The molecular weight between the crosslinks can be selected to control the release profile - groups 3.2A and 3.2B [Figure 18] The release profile can be controlled by selecting the molecular weight between the crosslinks - groups 3.2C and 3.2D [Figure 19A] The LDP:maPEG ratio can be used to control the release profile - groups 3.3A and 3.3B [Figure 19B] The LDP:maPEG ratio can be used to control the release profile - groups listed in Table 19AA [Figure 20] The LDP:maPEG ratio can be used to control the release profile - groups 3.3C and 3.3D [Figure 21] The LDP:maPEG ratio can be used to control the release profile - groups 3.3E and 3.3F [Figure 22] The LDP:maPEG ratio can be used to control the release profile - groups 3.3G and 3.3H [Diagram 23] The LDP:maPEG ratio can be used to control the release profile - groups 3.3I and 3.3J [Figure 24A] Adjustable number of days required for 100% release - 8% and 10% PEG at pH=8.0, 37℃ [Figure 24B] Adjustable number of days required for 100% release - 6% PEG at pH=8.0, 37℃ [Figure 24C] Adjustable number of days required for 100% release - 6% and 8% PEG at pH=8.0, 37℃ [Diagram 25] Adjustable number of days required for 100% release - Cumulative release of 6% maPEG with varying SS:SG ratio at pH=7.2 and 37°C - Groups 56-1, 56-2, 56-3, 54-4 and 54-3 [Figure 26] Adjustable number of days required for 100% release - Cumulative release of 8% maPEG with varying SS:SG ratios at pH=7.2 and 37°C - Groups 56-4, 56-5, 56-6 and 54-6 [Figure 27] If the sugar content is high, 100% will be released within about two days, regardless of other factors. [Figure 28] AAV release profile is reproducible at various doses [Figure 29A] AuNP release profile can be reproduced by AAV [Figure 29B] AuNP release profile can be reproduced by AAV [Figure 29C] Dv90 particle size of groups 1 and 2 as listed in Tables 27A and 27B [Figure 29D] Dv90 particle size can be used to control the release profile (Groups 1 and 2 in Tables 27A and 27B) [Figure 29E] The Dv90 particle size can be used to control the release profile (Group 3 using microemulsions and Group 1 as described in Tables 27A and 27B). [Diagram 30]In vivo AAV2 transduction and GFP expression by formulated implants on days 0, 4, 7, 10, 14, 17, 21, 24, and 28. Days listed from top to bottom should be read from left to right on the graph. [Diagram 31] (A) Diameter of fiber according to Example 5 over time, (B) Length of fiber according to Example 5 over time. Groups listed from top to bottom should be read from left to right on the graph. [Diagram 32] AAV2 Dose Comparison, Theoretical vs. PCR vs. ELISA - PCR and ELISA results are comparable. Groups listed from top to bottom should be read from left to right on the graph. [Diagram 33] AAV2 Fiber - Release Profile Used for In Vivo Administration with Example 5 [Figure 34A] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - Anterior chamber cells [Figure 34B] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - Anterior chamber cells [Figure 34C] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - Anterior chamber cells [Fig. 34D] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - Anterior chamber cells [Figure 34E] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - Anterior chamber cells [Figure 35A] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - anterior chamber flare [Figure 35B] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - anterior chamber flare [Figure 35C] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - anterior chamber flare [Figure 35D] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - anterior chamber flare [Figure 35E] In vivo administration of AAV2 implant according to Example 5: Inflammation scores - anterior chamber flare [Figure 36A] In vivo administration of AAV2 implants according to Example 5: Inflammation scores - vitreous cells [Figure 36B] In vivo administration of AAV2 implants according to Example 5: Inflammation scores - vitreous cells [Figure 36C] In vivo administration of AAV2 implants according to Example 5: Inflammation scores - vitreous cells [Figure 36D] In vivo administration of AAV2 implants according to Example 5: Inflammation scores - vitreous cells [Figure 36E] In vivo administration of AAV2 implants according to Example 5: Inflammation scores - vitreous cells [Figure 36F] In vivo administration of AAV2 implants according to Example 5: Inflammation scores - vitreous cells [Figure 37] In vivo administration of AAV2 implant according to Example 5: FAF images and inflammation scores for G1 placebo on days 2, 14, 29, 44, and 56. VH=vitreous opacification, VC=vitreous cells, AC=anterior chamber cells, and AF=anterior chamber flare. [Figure 38] In vivo administration of AAV2 implants according to Example 5: FAF images and inflammation scores of G2 AAV2 solutions on days 2, 14, 29, 44, and 56. VH = vitreous opacity, VC = vitreous cells, AC = anterior chamber cells, and AF = anterior chamber flare. [Figure 39] In vivo administration of AAV2 implants according to Example 5: FAF images and inflammation scores of G3 AAV2 implants on days 2, 14, 29, 44, and 56. VH=vitreous opacification, VC=vitreous cells, AC=anterior chamber cells, and AF=anterior chamber flare. [Diagram 40] In vivo administration of AAV2 implant according to Example 5: FAF images and inflammation scores of G4 AAV2 solution + TA on days 2, 14, 29, 44, and 56. VH = vitreous opacity, VC = vitreous cells, AC = anterior chamber cells, and AF = anterior chamber flare. [Diagram 41]In vivo administration of AAV2 implant according to Example 5: FAF images and inflammation scores of G5 AAV2 implant+TA on days 2, 14, 29, 44, and 56. VH=vitreous opacification, VC=vitreous cells, AC=anterior chamber cells, and AF=anterior chamber flare. [Diagram 42] In vivo administration of AAV2 implant according to Example 5: FAF images and inflammation scores of G6 AAV2 solution + TKI on days 2, 14, 29, 44, and 56. VH = vitreous opacity, VC = vitreous cells, AC = anterior chamber cells, and AF = anterior chamber flare. [Diagram 43] In vivo administration of AAV2 implant according to Example 5: FAF images and inflammation scores of G7 AAV2 implant + TKI on days 2, 14, 29, 44, and 56. VH = vitreous opacity, VC = vitreous cells, AC = anterior chamber cells, and AF = anterior chamber flare. [Diagram 44] In vivo biodistribution of AAV2 following AAV2 implant or AAV2 bolus according to Example 5: Bar graph showing AAV vector copy number in the aqueous and vitreous humor of rabbits administered an AAV2 bolus or an AAV implant, as indicated. [Diagram 45] (A) Diameter change over time of the fiber according to Example 6. (B) Length change over time of the fiber according to Example 6. [Figure 46] AAV2.7m8 Fiber Release Profile Used for In Vivo Administration According to Example 6 [Figure 47A] In vivo administration of AAV2.7m8 implant according to Example 6: Inflammation scores - Anterior chamber cells [Figure 47B] In vivo administration of AAV2.7m8 implant according to Example 6: Inflammation scores - anterior chamber flare [Figure 48] In vivo administration of AAV2.7m8 implant according to Example 6: FAF images and inflammation scores for the bolus, immediate release, and intermediate release groups on day 9. VH=vitreous opacity, VC=vitreous cells, AC=anterior chamber cells, AF=anterior chamber flare. [Figure 49]In vivo administration of AAV2.7m8 implant according to Example 6: FAF images and inflammation scores for the bolus, immediate release, and intermediate release groups on day 30. VH=vitreous opacity, VC=vitreous cells, AC=anterior chamber cells, and AF=anterior chamber flare. [Figure 50] In vivo administration of AAV2.7m8 implant according to Example 6: FAF images and inflammation scores for the bolus, immediate release, and intermediate release groups on day 72. VH=vitreous opacification, VC=vitreous cells, AC=anterior chamber cells, and AF=anterior chamber flare. [Figure 51] Time-dependent release of AAV2 and AAV2.7m8 based on ELISA results from implants used for in vivo administration according to Example 5 (first rabbit study) and Example 6 (second rabbit study) [Figure 52] Results of anti-drug antibody (ADA) assay against AAV2.7m8 in serum from bolus, fast release, and intermediate release groups from week 0 to week 13, according to Example 6 (second rabbit study). The sample titer is the highest dilution at which the sample's mean OD value is equal to or greater than the assay cutoff, and the next highest dilution is below the assay cutoff. Values ​​are expressed as inverse dilutions. [Figure 53] Vector shedding quantified as copies of heterologous nucleic acid sequence detected in plasma at days 0, 2, 4, 7, and 2 weeks post-dose in the bolus, immediate release, and intermediate release groups from Example 6 (second rabbit study). LLOQ = lower limit of quantification (5000VG / mL). [Figure 54] Serum ADA titers versus vector shedding in the bolus, fast-release, and intermediate-release groups from Example 6 (second rabbit study). Vector copy number per mL refers to copies of the heterologous nucleic acid sequence within the AAV (in this case eGFP). [Figure 55] GFP quantification results at 14 weeks in ocular tissues of the bolus, fast-release, and intermediate-release groups according to Example 6 (second rabbit study). [Figure 56]Vector copies in plasma on day 2 versus anterior chamber cell score (week 3) in the bolus, fast-release, and intermediate-release groups from Example 6 (second rabbit study). Vector copy number per mL refers to copies of the heterologous nucleic acid sequence within the AAV (in this case eGFP). [Figure 57A] In vivo administration of AAV2.7m8 implant according to Example 7: Inflammation score - total cells in the anterior chamber. * indicates treatment of an IVT dose of TA for ocular inflammation. [Figure 57B] In vivo administration of AAV2.7m8 implant according to Example 7: Inflammation scores - placebo. [Figure 57C] In vivo administration of AAV2.7m8 implants according to Example 7: Inflammation scores - bolus. [Fig. 57D] In vivo administration of AAV2.7m8 implants according to Example 7: Inflammation scores - rapid release. [Figure 57E] In vivo administration of AAV2.7m8 implants according to Example 7: Inflammation scores - intermediate release. [Figure 58] In vivo administration of AAV2.7m8 implant according to Example 7: Inflammation scores - anterior chamber flare in the bolus, immediate release and intermediate release groups. [Figure 59] In vivo administration of AAV2.7m8 implant according to Example 7: FAF images and inflammation scores for the bolus, immediate release, and intermediate release groups at week 8. VH=vitreous opacity, VC=vitreous cells, AC=anterior chamber cells, and AF=anterior chamber flare. [Figure 60] In vivo administration of AAV2.7m8 implant according to Example 7: FAF images and inflammation scores for the bolus, immediate release, and intermediate release groups at week 12. VH=vitreous opacity, VC=vitreous cells, AC=anterior chamber cells, and AF=anterior chamber flare. [Figure 61] Vector shedding quantified as copies of heterologous nucleic acid sequence (here eGFP) detected in plasma by qPCR at pre-dose, days 2, 4, 7, 10, weeks 2 and 3 post-dose in placebo, bolus, immediate release, and intermediate release groups from Example 7 (NHP study). LLOQ = lower limit of quantification (5000VG / mL). [Figure 62] In vitro release profiles of AAV2.7m8 from intermediate and fast release implants used in the NHP study according to Example 7 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Xerogel According to some aspects of the invention, a xerogel is a dehydrated gel comprising a matrix and / or polymer network comprising at least two covalently crosslinked multi-arm precursors. In some embodiments, a xerogel is a dehydrated organogel comprising a matrix and / or polymer network comprising at least two covalently crosslinked multi-arm precursors.

[0071] Thus, a precursor is always a "functional polymer" that can participate in a cross-linking reaction with another precursor to form a polymer network or matrix. Thus, the term "non-functional polymer" refers to a polymer that may be present in the organogel, xerogel, hydrogel and / or implant (or pharma- ceutically acceptable implant) of the present invention, but that does not participate in a cross-linking reaction with a precursor and does not form a polymer network or matrix.

[0072] The precursors used in the present invention can be any polymer as long as they are soluble in organic solvents, can react with other precursors, and are non-reactive with biological agents. The polymers can be selected from natural polymers, synthetic polymers, or biosynthetic polymers.

[0073] Natural polymers can include glycosaminoglycans, polysaccharides (eg, dextran), polyamino acids, and proteins, or mixtures or combinations thereof.

[0074] In some embodiments, synthetic precursors are preferred. Synthetic refers to molecules that do not occur in nature or that do not normally occur in the human body. Synthetic polymers can generally be any polymer that is synthetically produced by different types of polymerization, including free radical polymerization, anionic or cationic polymerization, chain growth or addition polymerization, condensation polymerization, ring-opening polymerization, etc. Polymerization can be initiated by some initiators, light and / or heat, or can be mediated by a catalyst.

[0075] Generally, for the purposes of the present invention, one or more synthetic polymers from the group containing one or more polyalkylene glycol units, such as polyethylene glycol (PEG), polypropylene glycol, poly(ethylene glycol)-block-poly(propylene glycol) copolymers, or polyethylene oxide, polypropylene oxide, polyvinyl alcohol, poly(vinylpyrrolidinone), polylactic acid, polylactic acid-co-glycolic acid, random or block copolymers, or any combination / mixtures thereof, can be used, although this list is not intended to be limiting.

[0076] The precursors have functional groups that react with each other. The functional groups are configured to react with each other in an electrophile-nucleophile reaction or to participate in other polymerization reactions. Thus, according to the present invention, each precursor contains at least one nucleophile or at least one electrophile.

[0077] Nucleophiles that can be used in the present invention can include amines, such as primary amines, thiols, azides, or hydrazides. In certain embodiments, at least one precursor includes a nucleophile, preferably a primary amine.

[0078] Electrophiles that can be used in the present invention may include succinimidyl esters, succinimidyl carbonates, nitrophenyl carbonates, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, dibenzocyclooctynes, norbornenes, epoxides, mesylates, tosylates, tresyls, cyanurates, orthopyridyl disulfides, or halides. These electrophiles may contain reactive groups that participate in electrophile-nucleophile reactions. For example, in one embodiment of the present invention, succinimidyl esters may contain reactive groups such as succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), or succinimidyl glutaramide.

[0079] The term "multi-arm" precursor means that the precursor is branched. In the case of a multi-arm polymer, the core refers to a continuous portion of the molecule joined to arms extending from the core, the arms often having nucleophiles or electrophiles at the ends of the branches. Note that the precursor may have, for example, 2-100 arms, each arm having a terminus, and some precursors may be dendrimers or other highly branched materials. The arms of the precursor refer to linear chains of chemical groups connecting the crosslinkable groups to the polymer core. Some embodiments are precursors having 3-300 arms. One of ordinary skill in the art will readily appreciate that all ranges and values ​​within the explicitly stated ranges are contemplated, for example, 4, 6, 8, 10, 12, 4-16, 8-100, 6, 8, 10, 12, or at least 4 arms.

[0080] In some embodiments, when each precursor is multi-armed, it contains two or more arms, and thus two or more of the same or different electrophiles or nucleophiles, such that each nucleophile can react with another electrophile (within the same precursor or in another precursor) in an electrophile-nucleophile reaction to form a crosslinked polymer product. Thus, for example, in some embodiments, the precursor has four arms, with each arm terminating in either a nucleophile or electrophile, which may be the same or different from the other arms.

[0081] According to one aspect of the invention, the xerogel comprises at least two multi-arm precursors, the first multi-arm precursor comprising a nucleophile and / or an electrophile, and the second multi-arm precursor comprising a nucleophile and / or an electrophile. In this embodiment, the first multi-arm precursor and the second multi-arm precursor are covalently crosslinked to each other in an electrophile-nucleophile reaction. In this context, multi-arm refers to at least 10 arms, at least 8 arms, for example at least 4 arms.

[0082] In one embodiment, when the xerogel comprises two multi-arm precursors, it can comprise a first multi-arm precursor comprising a nucleophile, such as an amine, such as a primary amine, a thiol, an azide or a hydrazide, and a second multi-arm precursor comprising an electrophile, such as a succinimidyl ester, a succinimidyl carbonate, a nitrophenyl carbonate, an aldehyde, a ketone, an acrylate, an acrylamide, a maleimide, a vinyl sulfone, an iodoacetamide, an alkene, an alkyne, a dibenzocyclooctyne, a norbornene, an epoxide, a mesylate, a tosylate, a tresyl, a cyanurate, an orthopyridyl disulfide, or a halide. The nucleophile and the electrophile are covalently crosslinked to each other in an electrophile-nucleophile reaction. In some embodiments, the first multi-arm precursor is a primary amine and the second multi-arm precursor is a succinimidyl ester.

[0083] According to the present invention, the xerogel comprises at least three multi-arm precursors, including a first multi-arm precursor comprising a nucleophile and / or electrophile, a second multi-arm precursor comprising a nucleophile and / or electrophile, and a third multi-arm precursor comprising a nucleophile and / or electrophile. In this embodiment, the first multi-arm precursor, the second multi-arm precursor, and the third multi-arm precursor are covalently crosslinked to each other in an electrophile-nucleophile reaction. In this context, multi-arm refers to at least 10 arms, at least 8 arms, for example at least 4 arms.

[0084] According to the present invention, the xerogel comprises at least three multi-arm precursors, including a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and a third multi-arm precursor comprising an electrophile. In this embodiment, the first multi-arm precursor and the second multi-arm precursor, and the first multi-arm precursor and the third multi-arm precursor, are covalently crosslinked to each other in an electrophile-nucleophile reaction. In this context, multi-arm refers to at least 10 arms, at least 8 arms, for example at least 4 arms. In this embodiment, the nucleophile may be an amine, such as a primary amine, a thiol, an azide, or a hydrazide, and the electrophile may be a succinimidyl ester, succinimidyl carbonate, nitrophenyl carbonate, an aldehyde, a ketone, an acrylate, an acrylamide, a maleimide, a vinyl sulfone, an iodoacetamide, an alkene, an alkyne, a dibenzocyclooctyne, a norbornene, an epoxide, a mesylate, a tosylate, a tresyl, a cyanurate, an orthopyridyl disulfide, or a halide. For example, in one embodiment of the invention, the succinimidyl ester may contain a reactive group such as succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), or succinimidyl glutaramide.

[0085] Some precursors may have a longer hydrolysis half-life than other precursors. That is, they may require a longer time to decompose. This may be due in part to the reactive groups contained in the precursor. For example, a PEG polymer containing an electrophilic group such as a succinimidyl ester group containing a reactive group such as succinimidyl succinate (SS) will have a shorter hydrolysis half-life than a PEG polymer containing an electrophilic group such as a succinimidyl ester group containing a reactive group such as succinimidyl glutarate (SG). For example, a PEG polymer containing an electrophilic group such as a succinimidyl ester group containing a reactive group such as succinimidyl succinate (SG) will have a shorter hydrolysis half-life than a PEG polymer containing an electrophilic group such as a succinimidyl ester group containing a reactive group such as succinimidyl glutarate (SAP). For example, PEG polymers containing electrophilic groups such as succinimidyl ester groups that contain a reactive group such as succinimidyl succinate (SAP) will have a reduced hydrolysis half-life compared to PEG polymers containing electrophilic groups such as succinimidyl ester groups that contain a reactive group such as succinimidyl glutarate (SAZ).

[0086] According to the present invention, when the xerogel comprises three multi-arm precursors, the xerogel may comprise a first multi-arm precursor comprising a nucleophile such as an amine, a second multi-arm precursor comprising an electrophile such as a succinimidyl ester, and a third precursor comprising an electrophile that may or may not be a succinimidyl ester. Thus, in another embodiment, when the xerogel comprises three precursors, it may comprise a first multi-arm precursor comprising a nucleophile such as a primary amine, a second multi-arm precursor comprising an electrophile such as a succinimidyl ester comprising a first reactive group, and a third precursor comprising an electrophile that is a succinimidyl ester comprising a second reactive group. In this embodiment, the reactive group is selected from succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), or succinimidyl azelate (SAZ).

[0087] In some embodiments, the precursor is a polyethylene glycol precursor. Thus, in some embodiments, the covalently crosslinked polymer network or matrix of precursors is made from a polyethylene glycol-containing precursor. Polyethylene glycol (PEG, also called polyethylene oxide) refers to a polymer with repeating groups (CH2CH20)n, where n is at least 3.

[0088] Thus, polymer precursors having polyethylene glycol have at least three of these repeating groups linearly linked to each other. PEG polymers that terminate with hydroxyl or methoxy groups that do not participate in the crosslinking reaction between precursors are referred to as "non-functional PEGs" as described hereinabove, and therefore are not used as one of the precursors. Thus, PEG polymers that terminate with a nucleophile selected from primary amines, thiols, azides, or hydrazides are considered to be "functional PEGs" and can be used as one of the precursors. Furthermore, PEG polymers that terminate with an electrophile selected from succinimidyl esters, succinimidyl carbonates, nitrophenyl carbonates, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, dibenzocyclooctynes, norbornenes, epoxides, mesylates, tosylates, tresyls, cyanurates, orthopyridyl disulfides, or halides are considered to be "functional PEGs" and can be used as one of the precursors.

[0089] The polymer network of the hydrogel implants of the invention can include one or more multi-arm PEG units having 2-10 arms, or 4-8 arms, or 4, 5, 6, 7, or 8 arms. The PEG units can have different or the same number of arms. In certain embodiments, the PEG units used in the hydrogels of the invention have 4 and / or 8 arms. In certain particular embodiments, a combination of 4-armed and 8-armed PEG units is utilized.

[0090] In certain embodiments of the present invention, the polyethylene glycol units used as precursors have an average molecular weight in the range of about 2,000 to about 100,000 daltons, or in the range of about 10,000 to about 60,000 daltons, or in the range of about 15,000 to about 50,000 daltons. In certain specific embodiments, the polyethylene glycol units have an average molecular weight in the range of about 10,000 to about 40,000 daltons, or about 20,000 daltons. PEG precursors of the same average molecular weight may be used, or PEG precursors of different average molecular weights may be combined with each other. The average molecular weight of the PEG precursors used in the present invention is given as a number average molecular weight (Mn), which in certain embodiments may be determined by MALDI.

[0091] In a 4-arm PEG, each arm can have an average arm length (or molecular weight) that is the total molecular weight of PEG divided by 4. Thus, one precursor that can be used in the present invention, the 4a20k PEG precursor, has four arms with an average molecular weight of about 5,000 daltons each. In addition to the 4a20k PEG precursor, the present invention can also use the 8a20k PEG precursor, which has eight arms with an average molecular weight of 2,500 daltons each.

[0092] When referring to a PEG precursor having a particular average molecular weight, such as a 15k PEG or 20k PEG precursor, the indicated average molecular weight (i.e., Mn of 15,000 or 20,000, respectively) refers to the PEG portion of the precursor before end groups are added (where "20k" means 20,000 Daltons and "15k" means 15,000 Daltons; the same abbreviations are used herein for other average molecular weights of PEG precursors). In certain embodiments, the Mn of the precursor PEG portion is determined by MALDI. The degree of substitution with end groups disclosed herein may be determined by H-NMR after end group functionalization.

[0093] In various embodiments of the invention, the xerogel comprises at least two multi-arm precursors, the first of which is a multi-arm PEG precursor comprising a nucleophile, such as an amine, e.g., a primary amine, and in this embodiment, the second of which is a multi-arm PEG precursor comprising an electrophile, such as a succinimidyl ester.

[0094] In various embodiments of the present invention, the xerogel comprises three multi-arm precursors, the first multi-arm precursor being a multi-arm PEG precursor comprising a nucleophile such as an amine, e.g., a primary amine. In this embodiment, the second multi-arm precursor is a multi-arm PEG precursor comprising an electrophile such as a succinimidyl ester comprising a first reactive group. In this embodiment, the third multi-arm precursor is a multi-arm PEG precursor comprising an electrophile such as a succinimidyl ester comprising a second reactive group. In this embodiment, the first reactive group and the second reactive group can be selected from succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), or succinimidyl azelate (SAZ). SS, SG, SAP, and SAZ are all succinimidyl ester reactive groups with ester groups that degrade by hydrolysis in water. In some embodiments, the first multi-arm precursor is succinimidyl succinate (SS) and the second multi-arm precursor is succinimidyl glutarate (SG).

[0095] Each and any combination of PEG precursors containing electrophilic and nucleophilic groups disclosed herein can be used to prepare implants according to the present invention.For example, any 4-arm or 8-arm PEG precursor (e.g., with succinimidyl esters containing SS, SG, SAP, or SAZ reactive groups) can be combined with any 4-arm or 8-arm PEG precursor (e.g., with NH2 groups or another nucleophile).Furthermore, the PEG units of the electrophilic and nucleophilic precursors can have the same average molecular weight or different average molecular weights.

[0096] One such combination is a PEG amine precursor and two PEG succinimidyl ester precursors, one containing a SS reactive group and the other containing a SG reactive group. The inventors have discovered that by keeping the molar ratio of PEG amine to PEG succinimidyl ester at 1:1 and varying the molar ratio of the succinimidyl ester SS and SG reactive groups, they can control the time it takes for the polymer network to degrade in aqueous solution under physiological conditions. The amount of PEG SS and SG to be used to reach a particular molar ratio of the two reactive groups can be calculated by one skilled in the art and can be explained as follows:

[0097] The amount of PEG amine and PEG ester (SS and SG) to be used is calculated by the stoichiometric formula of the molar ratio and the conversion of moles to grams. First, the molar ratio of reactive end groups between amine, succinimidyl succinate, and succinimidyl glutarate is determined. In one example formulation, 4a20k PEG NH2, 4a20k PEG SS, and 4a40k PEG SG are used. The molar ratio of amine to succinimidyl ester group is 1:1, and the molar ratio of SS to SG is 80:20. The final end group molar ratio between 4a20k NH2:4a20k SS:4a40k SG is 1.0:0.8:0.2. Then, the mass is determined using the stoichiometric conversion of grams to moles and vice versa. Below is an example calculation of 4a20k SS at the above molar ratio using 100g of 4a20k NH2.

number

[0098] Alternatively, the amount of PEG can be determined by calculating the "molecular weight between bridges" (MWc) and arm length ratio. MWc can be calculated through the sum of the average arm lengths of each multi-arm PEG precursor.

number

[0099] The arm length ratio is calculated by dividing the PEG arm length by the MWc. The amount of multi-arm precursor can be determined by multiplying the arm length ratio of a particular multi-arm precursor by the total PEG batch size. Below is an example calculation of the amount of 4a20k PEG SS for a total batch size of 100 g PEG:

number

[0100] In certain preferred embodiments, 4-arm PEG having an average molecular weight of about 20,000 daltons, and 4-arm PEG having an average molecular weight of about 40,000 daltons can be used to form polymer networks, and thus xerogels, in accordance with the present invention.

[0101] Thus, the first precursor, the second precursor, and / or the third precursor may be a 4a20k precursor, where 4 represents an arm and 20k represents Mn. Thus, for example, the first precursor, the second precursor, and / or the third precursor may be a 4a40k precursor. Thus, for example, the first precursor and / or the second precursor may be a 4a20k precursor and the third precursor may be a 4a40k precursor.

[0102] In certain embodiments, the nucleophile-containing crosslinker may be linked or conjugated to a visualization agent. A visualization agent is an agent that includes a fluorescent group or other group that allows visualization. For example, fluorophores such as fluorescein, rhodamine, coumarin, and cyanine may be used as visualization agents. The visualization agent may be conjugated to the crosslinker, for example, via a portion of the nucleophile of the crosslinker. Since a sufficient amount of nucleophile is required for crosslinking, "conjugated" or "conjugated" generally includes partial conjugation, meaning that only a portion of the nucleophile is used for conjugation with the visualization agent, such as about 1% to about 20%, or about 5% to about 10%, or about 8% of the nucleophile of the crosslinker may be conjugated with the visualization agent. In other embodiments, the visualization agent may be conjugated to the polymer precursor, for example, via a specific reactive species (such as an electrophile) of the polymer precursor.

[0103] Active Agent: Biologics The active agents according to the present invention may be multiple of the same biologic, or multiple different biologics. The biologic may be, for example, a polypeptide, a protein encapsulating a nucleic acid, a virus, or a lipid encapsulating a nucleic acid.

[0104] As used herein, a peptide is any compound that contains two or more amino acid residues joined by an amide bond formed between the carboxyl group of one amino acid residue and the amino group of the adjacent amino acid residue. The amino acid residues may be in the L- or D-form, natural or synthetic, linear as well as cyclic. The meaning of peptide also includes polypeptides and peptide dimers, which may be peptides linked C-terminus to N-terminus (tandem repeats) or C-terminus to C-terminus (parallel repeats).

[0105] A protein fragment is any section of a polypeptide sequence that is separated from the remainder of the protein and that takes on a primary, secondary, or tertiary structure of its own. In some embodiments, these fragments are at least 8 amino acids in length and are at least 40%-99% identical to the reference protein, more preferably 70%, 80%, or 90%, or 99% identical to the reference protein.

[0106] The structure of a protein or polypeptide is usually described by a primary structure, a secondary structure, a tertiary structure, and a quaternary structure. The amino acid sequence of a protein defines the primary structure. Thus, according to the present invention, a biologic may be a polypeptide comprising a primary structure.

[0107] Proteins rarely form random coils, and the high degree of specificity of their functions depends on the defined conformation of the polypeptide chain in the secondary structure. The most common types of secondary structure are alpha helices and beta sheets. Thus, according to the present invention, the biologic may be a polypeptide that contains a secondary structure.

[0108] The elements of secondary structure may be connected through various types of loops and turns into larger tertiary structures. The tertiary structure of a polypeptide is the three-dimensional shape of the protein. The tertiary structure will include a single polypeptide chain "backbone" with one or more secondary structures. The interactions and bonds of side chains within a particular protein determine its tertiary structure. Thus, according to the present invention, a biologic may be a polypeptide that includes a tertiary structure.

[0109] The quaternary structure of a protein is a close-packed arrangement of several polypeptide chains or subunits. Each of the subunits has its own primary, secondary, and tertiary structure. The subunits are held together by hydrogen bonds and van der Waals forces between non-polar side chains. Thus, according to the present invention, the biologic may be a polypeptide that includes a quaternary structure.

[0110] In some embodiments, a biologic according to the present invention is a recombinant protein or recombinant polypeptide, which are used interchangeably herein. In some embodiments, a recombinant protein refers to an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0111] The term "antibody(s)" as used herein refers to monoclonal or polyclonal antibodies. The term "antibody(s)" as used herein includes, but is not limited to, recombinant antibodies produced by recombinant techniques known in the art. "Antibody(s)" includes antibodies of any species, particularly mammalian species, such as human antibodies of any isotype including IgA1, IgA2, IgD, IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE and IgM and modified variants thereof, non-human primate antibodies, such as from chimpanzees, baboons, rhesus monkeys or cynomolgus monkeys, rodent antibodies, such as from mice, rats or rabbits, goat or horse antibodies, as well as camelid antibodies (e.g., antibodies from camels or llamas, such as nanobodies) and derivatives thereof, or antibodies of avian species, such as chicken antibodies, or fish species, such as shark antibodies. The term "antibody(s)" also refers to "chimeric" antibodies in which a first portion of at least one heavy and / or light chain antibody sequence is derived from a first species and a second portion of the heavy and / or light chain antibody sequence is derived from a second species. The term "antibody(s)" also includes antigen-binding portions or antigen-binding fragments of antibodies. The term "antigen-binding fragment" also refers to antibodies that contain at least one heavy or light chain immunoglobulin domain as known in the art and bind to one or more antigen(s). Examples of antibody fragments that may be used as biologics include Fab, Fab', F(ab')2, and Fv and scFv fragments, as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies, single chain antibodies, bispecific antibodies, triabodies, tetraspecific antibodies, or multispecific antibodies (including but not limited to Fab-Fv constructs) formed from antibody fragments or antibodies. In some embodiments, the antibody is a vNAR, a camelid antibody, a VHH antibody, or an antigen-binding portion thereof. Antibody fragments as defined above are known in the art.

[0112] A fusion protein is a protein composed of at least two domains encoded by separate genes that are joined together so that they are transcribed and translated as a single unit to produce a single polypeptide. Fusion protein is sometimes used interchangeably with chimeric protein.

[0113] As used herein, the term "cytokine" refers to a molecule that regulates cell-cell interactions in an immune or inflammatory response. Cytokines include, but are not limited to, monokines and lymphokines.

[0114] "Hormones" as used herein refer to any signaling molecule that affects a particular cell type. The term "hormones" encompasses all types of hormones, including endocrine, paracrine, autocrine, and endocrine hormones. In some embodiments, hormones as used herein refer to polypeptide hormones.

[0115] "Transcription factor" as used herein should be interpreted in the broadest possible sense as any protein involved in the process of converting DNA into RNA, i.e., transcribing. Transcription factors include a variety of proteins that initiate and control the transcription of genes. One distinguishing feature of transcription factors is that they possess a DNA-binding domain that confers the ability to bind to specific sequences of DNA, called enhancer or promoter sequences. Some transcription factors bind to DNA promoter sequences near the transcription start site and aid in the formation of a transcription initiation complex. Other transcription factors bind to regulatory sequences, such as enhancer sequences, and may stimulate or repress the transcription of the associated gene.

[0116] General examples of recombinant proteins that may be considered as biologics for inclusion in the pharma- ceutically acceptable implants of the present invention include RPE65, REP1, RPGR, BEST1, anti-VEGF inhibitors such as aflibercept, ranibizumab, brolucizumab, or bevacizumab, 31-tracta sodium, adalimumab, infliximab, hRS1, hCNGB3, ABCR, MYO7A, endostatin, angiostatin, TNF[α] receptor, TGF[β]2 receptor, IRS-1, IGF-1, angiogenin, angiopoietin-1, DeM, acidic or basic fibroblast growth factor (aFGF and bFGF), FGF-2, follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), scatter factor (SF), leptin, midkine, placental growth factor (PGF), platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN), RdCVF (rod-derived cone survival factor), progranulin, proliferin, transforming growth factor-α (TGF-α), PEDF, transforming growth factor-β (TGF-β) ), vascular permeability factor (VPF), CNTF, BDNF, GDNF, PEDF, NT3, BFGF, ephrin, EPO, NGF, GMF, aFGF, NT5, Gax, growth hormone, [α]-1-antitrypsin, calcitonin, leptin, apolipoproteins, vitamins, enzymes in the synthesis of hormones or neurotransmitters, chemokines, cytokines such as IL-1, IL-8, IL-10, IL-12, IL-13, and their receptors, antibodies blocking any one of the above receptors, TIMPs (TIMP-1, TIMP-2, TIMP-3, TIMP-4), etc.), angioarrestin, endostatin, e.g., endostatin XVIII and endostatin XV, ATF, fusion protein of endostatin and angiostatin, C-terminal hemopexin domain of matrix metalloproteinase-2, kringle 5 domain of human plasminogen, fusion protein of endostatin and kringle 5 domain of human plasminogen, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prolactin fragment, proliferin-related protein (PRP),Antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59 complement fragment, C3a and C5a inhibitors, combined attack membrane inhibitor, factor H, ICAM, VCAM, caveolin, PKC zeta, junction proteins, JAM, CD36, MERTK, vasculostatin, vasostatin (calreticulin fragment), thrombospondin, fibronectin, especially fibronectin fragment gro-beta, heparinase, human chorionic gonadotropin (hCG), interferon alpha / beta / gamma, interferon-inducible protein (IP -10), monokine induced by interferon gamma (Mig), interferon alpha-inducible protein 10 (IP10), fusion protein of Mig and IP10, soluble Fms-like tyrosine kinase 1 (FLT-1) receptor, kinase insert domain receptor (KDR), regulators of apoptosis such as Bcl-2, Bad, Bak, Bax, Bik, BcI-X short isoform and Gax, alpha 1 antitrypsin, factor IX, factor VIII, Cl-esterase inhibitor, β-globin or γ-globin, but are not limited thereto. The recombinant protein may also be a Cas9 polypeptide, a zinc finger nuclease, a TALEN polypeptide, or any combination thereof.

[0117] The biologic may be a lipid encapsulating a nucleic acid. The nucleic acid may be any nucleic acid selected from DNA and RNA. Some examples include ssDNA (single-stranded DNA), dsDNA (double-stranded DNA), plasmid DNA, diploid RNA, small interfering RNA (siRNA), microRNA, dsRNA, mRNA, lncRNA, piRNA, rmRNA, sRNA, tiRNA, eRNA, snoRNA, snRNA, circRNA (circular RNA), microRNA, long non-coding RNA, RNA aptamer, antisense oligonucleotide, guide RNA, tRNA, or any combination thereof. In some embodiments, the lipid encapsulating the nucleic acid is in the form of a lipid nanoparticle. Lipid nanoparticles are spherical vesicles made of ionizable lipids that are positively charged at low pH (allowing nucleic acid complexation) and neutral at physiological pH. In some embodiments, the nucleic acid is encapsulated in a microvesicle, nanovesicle, exosome, or endosome.

[0118] Biological agents may include viruses such as retroviruses, adenoviruses, adeno-associated viruses (AAV), lentiviruses, and herpes simplex viruses. Most viruses contain at least an outer protein shell and endogenous nucleic acid, so the term "virus" can also be used interchangeably with the term "protein(s) encapsulating nucleic acid". In some embodiments, when a biological agent is a virus, it contains not only its endogenous nucleic acid but also heterologous nucleic acid. Heterologous nucleic acid is any nucleic acid that does not belong to a virus.

[0119] The nucleic acid may be DNA or RNA. The heterologous nucleic acid may take the form of ssDNA (single-stranded DNA), dsDNA (double-stranded DNA), plasmid DNA, diploid RNA, small interfering RNA (siRNA), microRNA, dsRNA, mRNA, lncRNA, piRNA, rmRNA, sRNA, tiRNA, eRNA, snoRNA, snRNA, circRNA (circular RNA), microRNA, long non-coding RNA, RNA aptamer, antisense oligonucleotide, guide RNA, tRNA, or any combination thereof.

[0120] The heterologous nucleic acid may be a coding or non-coding nucleic acid. In certain embodiments, the heterologous nucleic acid(s) encodes a therapeutic protein that is not present in the subject, or that is present in the subject but at a reduced level compared to healthy subjects.

[0121] When the heterologous nucleic acid is a non-coding nucleic acid, it may be selected from the group consisting of ssDNA (single-stranded DNA), dsDNA (double-stranded DNA), small interfering RNA (siRNA), microRNA, dsRNA, lncRNA, piRNA, rmRNA, sRNA, tiRNA, eRNA, snoRNA, snRNA, circRNA (circular RNA), RNA aptamer, antisense oligonucleotide, guide RNA, tRNA, or any combination thereof.

[0122] When the heterologous nucleic acid is a coding nucleic acid, it preferably codes for a therapeutic protein. In some embodiments, the therapeutic protein is RPE65, REP1, RPGR, BEST1, anti-VEGF inhibitors such as aflibercept, ranibizumab, brolucizumab, or bevacizumab, 33-tract 33 sodium, adalimumab, infliximab, hRS1, hCNGB3, ABCR, MYO7A, endostatin, angiostatin, TNF[α] receptor, TGF[β]2 receptor, IRS-1, IGF-1, angiogenin, angiopoietin-1, DeM, acidic or basic fibroblast growth factor (AF). GF and bFGF), FGF-2, follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), scatter factor (SF), leptin, midkine, placental growth factor (PGF), platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN), RdCVF (rod-derived cone survival factor), progranulin, proliferin, transforming growth factor-α (TGF-α), PEDF, transforming growth factor-β (TGF-β), vascular permeability factor (VPF), C NTF, BDNF, GDNF, PEDF, NT3, BFGF, ephrin, EPO, NGF, GMF, aFGF, NT5, Gax, growth hormone, [α]-1-antitrypsin, calcitonin, leptin, apolipoproteins, vitamins, enzymes in the biosynthesis of hormones or neurotransmitters, chemokines, cytokines such as IL-1, IL-8, IL-10, IL-12, IL-13 and their receptors, antibodies blocking any one of said receptors, TIMPs, e.g. TIMP-1, TIMP-2, TIMP-3, TIMP-4, angiotensin II receptors, arrestin, endostatin, e.g., endostatin XVIII and endostatin XV, ATF, fusion protein of endostatin and angiostatin, C-terminal hemopexin domain of matrix metalloproteinase-2, kringle 5 domain of human plasminogen, fusion protein of endostatin and kringle 5 domain of human plasminogen, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prolactin fragment, proliferin-related protein (PRP),Antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59 complement fragment, C3a and C5a inhibitors, combined attack membrane inhibitor, factor H, ICAM, VCAM, caveolin, PKC zeta, junction proteins, JAM, CD36, MERTK, vasculostatin, vasostatin (calreticulin fragment), thrombospondin, fibronectin, especially fibronectin fragment gro-beta, heparinase, human chorionic gonadotropin (hCG), interferon alpha / beta / gamma, interferon-inducible protein (I P-10), monokine induced by interferon gamma (Mig), interferon alpha-inducible protein 10 (IP10), fusion protein of Mig and IP10, soluble Fms-like tyrosine kinase 1 (FLT-1) receptor, kinase insert domain receptor (KDR), regulators of apoptosis such as Bcl-2, Bad, Bak, Bax, Bik, Bcl-X short isoform and Gax, alpha 1 antitrypsin, factor IX, factor VIII, Cl-esterase inhibitors, β-globin or γ-globin, but are not limited to these. In some embodiments, the therapeutic protein is RPE65, REP1, RPGR, BEST1, anti-VEGF inhibitors such as aflibercept, ranibizumab, or bevacizumab, hRS1, hCNGB3, ABCR, MYO7A, endostatin, angiostatin.

[0123] In some embodiments, the biologic is a virus, and the virus is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, such as AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In other preferred embodiments, the virus is AAV2, AAV2.7m8, or AAV8. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding nucleic acid sequence or a non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can code for a therapeutic protein as described herein above.

[0124] The total amount or concentration of a biologic contained in a pharma- ceutically acceptable implant varies depending on the type of biologic.

[0125] In one embodiment, the biologic is a virus and is administered in a pharma- ceutically acceptable implant at least 10 9 In certain embodiments, the virus is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 9 ~10 15 In another preferred embodiment, the virus is present in a pharma- ceutical implant in a total amount of 10 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 In some embodiments, the virus is present in a total amount of at least 10 vg. 13 vg / cm 3 , e.g. at least 10 14 vg / cm 3 is contained in the pharma- ceutically acceptable implant at a total concentration of

[0126] In one embodiment, the biologic is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, and is administered in a pharmacologic implant in a concentration of at least 10 9 In certain embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 9 ~10 15 In another preferred embodiment, the AAV is present in a pharma- ceutical implant in a total amount of 10 vg. 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 In some embodiments, the AAV comprises at least 10 13 vg / cm 3 , e.g. at least 10 14 vg / cm 3 is contained in the pharma- ceutically acceptable implant at a total concentration of

[0127] In one embodiment, the biologic is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, and is administered in a pharmacologic implant in a concentration of at least 10 10 In certain embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 10 ~10 15 In another preferred embodiment, the AAV is present in a pharma- ceutical implant in a total amount of 10 vg. 10 ~10 15 vg, or 10 10 ~10 14 vg, or 10 10 ~10 13 vg, or 1010 ~10 12 vg, or 10 10 ~10 11 In some embodiments, the AAV comprises at least 10 13 vg / cm 3 , e.g. at least 10 14 vg / cm 3 is contained in the pharma- ceutically acceptable implant at a total concentration of

[0128] In some embodiments, the AAV is administered in a 10 10 ~10 15 In some embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 10 ~10 14 In some embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 10 ~10 13 In some embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 10 ~10 12 In some embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 10 ~10 11 Contains the total amount of vg.

[0129] In one embodiment, the biologic is a recombinant protein and is present in the pharma- ceutically acceptable implant in a total amount of at least 10 μg. In certain embodiments, the recombinant protein is present in the pharma-ceutically acceptable implant in a total amount of 10-3000 μg. In other preferred embodiments, the recombinant protein is present in the pharma-ceutically acceptable implant in a total amount of 10-2,500 μg, or 10-2000 μg.

[0130] In one embodiment, the biologic is an antibody and is present in the pharmaceutically acceptable implant in a total amount of at least 100 μg. In a particular embodiment, the antibody is present in the pharmaceutically acceptable implant in a total amount of 100-3000 μg. In another preferred embodiment, the antibody is present in the pharmaceutically acceptable implant in a total amount of 300-3000 μg.

[0131] In one embodiment, the biologic is an anti-VEGF antibody, such as ranibizumab, and is present in the pharmaceutically acceptable implant in a total amount of at least 500 μg. In a particular embodiment, the ranibizumab is present in the pharmaceutically acceptable implant in a total amount of 500-1000 μg. In another preferred embodiment, the ranibizumab is present in the pharmaceutically acceptable implant in a total amount of 300-1000 μg.

[0132] In one embodiment, the biologic is an anti-VEGF antibody, such as bevacizumab, and is present in the pharmaceutically acceptable implant in a total amount of at least 1,500 μg. In certain embodiments, the bevacizumab is present in the pharmaceutically acceptable implant in a total amount of 1,500-3,000 μg. In other preferred embodiments, the bevacizumab is present in the pharmaceutically acceptable implant in a total amount of 1,500-2,000 μg, or 1,250 μg.

[0133] In one embodiment, the biologic is a fusion protein such as aflibercept, and is included in the pharmaceutically acceptable implant in a total amount of at least 2000 μg. In a particular embodiment, the aflibercept is included in the pharmaceutically acceptable implant in a total amount of 2,000-3,000 μg. In another preferred embodiment, the aflibercept is included in the pharmaceutically acceptable implant in a total amount of 2,000 μg.

[0134] Dehydration stabilizer A dehydration stabilizer is an excipient and / or additive that protects and stabilizes a biological product or (non-biological product, including biological products) in a dry form or in an essentially moisture-free environment. A dehydration stabilizer is an excipient and / or additive that protects and stabilizes a biological product from damage. The term "protects a biological product from damage" means that the dehydration stabilizer prevents the biological product from denaturing, flocculating or agglomerating, thereby maintaining its functional activity. Thus, a dehydration stabilizer according to the present invention is an excipient and / or additive that maintains the structure and / or functional activity of a biological product. Thus, a biological product is considered to have maintained its structure and / or functional activity if it retains about 90%, such as about 80%, 70%, 60%, or at least 50% of its functional activity as measured by an appropriate analytical method.

[0135] Such analytical methods are known in the art. For example, if the biologic is a virus that contains a heterologous nucleic acid encoding a marker protein, such as green fluorescent protein (GFP), and said virus is subjected to a dehydration step, the skilled artisan can evaluate the functional activity of the virus by evaluating the infectivity, commonly known as the transduction efficiency of said virus. One such method involves mixing said virus with one or more dehydration stabilizers, and then subjecting particles containing a mixture of said dehydration stabilizers and virus to one or more dehydration steps. The particles can then be used to infect cells in vitro, and the transduction efficiency of the virus can be evaluated by the expression of the marker protein. In this way, the skilled artisan can conclude that the dehydration stabilizer was able to maintain the functional activity of the biologic, in this case the virus, by about 90%, for example about 80%, 70%, 60%, or at least 50%. Detailed methods of how the dehydration stabilizer can maintain the functional activity of the biologic are also described in the examples.

[0136] In another example, when the biologic is an antibody, the method includes mixing the antibody with one or more dehydration stabilizers, and then subjecting the particles containing the mixture of the dehydration stabilizers and the antibody to one or more dehydration steps.Thereafter, the skilled artisan can evaluate the functional activity of the antibody by various analytical methods known in the art.One of the powerful methods for quantitative and / or qualitative evaluation of such antibodies is the enzyme-linked immunosorbent assay (ELISA).

[0137] The term "dehydration stabilizer" should not be construed as being limited to a particular dehydration step or process, such as a lyophilization process. Instead, the present inventors have discovered that the dehydration stabilizer can protect a biologic from damage during any dehydration process. Such dehydration steps include, but are not limited to, lyophilization, spray drying, sterilization, and exposure to organic solvents, such as when forming organogels.

[0138] Additionally, the term "dehydration stabilizer" should not be construed as being limited to the dehydration process. Instead, the present inventors have discovered that the dehydration stabilizer can protect the biologic from damage even after the dehydration step and / or process is complete and the biologic remains in a dehydrated form for a period of time.

[0139] The total concentration of one or more dehydration stabilizers mixed with the biologic before the biologic is exposed to an organic solvent may be 5 mg / mL or more. Accordingly, the inventors have found that such lower total concentrations of one or more dehydration stabilizers can be used. The inventors have found that the total concentration of one or more dehydration stabilizers mixed with the biologic before subjecting the biologic to one or more dehydration steps can be 200 mg / mL to 5 mg / mL, 100 to 5 mg / mL, 55 mg / mL to 5 mg / mL, 85 mg / mL to 5 mg / mL, 30 mg / mL to 5 mg / mL, or about 5 mg / mL. The inventors have found that these concentrations are sufficient to protect the biologic from damage. In this context, the dehydration step can be any one of, or a combination of, freeze drying, spray drying, sterilization, and exposure to an organic solvent, such as in forming an organogel.

[0140] The total concentration of one or more dehydration stabilizers mixed with the biologic before the biologic is exposed to the organic solvent may be 5 mg / mL or more. Thus, the inventors have discovered that lower total concentrations of one or more dehydration stabilizers can be used. Thus, the total concentration of one or more dehydration stabilizers mixed with the biologic before the biologic is exposed to the organic solvent may be 200-5 mg / mL, 100-5 mg / mL, 85-5 mg / mL, 55-5 mg / mL, 30-5 mg / mL, or about 5 mg / mL. This concentration is sufficient to protect the biologic from damage in the process of directly exposing the biologic to the organic solvent. In this regard, the biologic is substantially insoluble in the organic solvent. The organic solvent may be any organic solvent that is carbon-based and liquid at room temperature and pressure. Such organic solvents may include methylene chloride, dimethyl carbonate, acetone, acetonitrile, ethyl acetate, and tetrahydrofuran. In some embodiments, the organic solvent is dimethyl carbonate. The term "substantially insoluble" generally refers to a solubility of 0.1 mg / mL or less, such as 0.01 mg / mL or less, 0.001 mg / mL or less, etc.

[0141] Below, several classes of dehydration stabilizers are described. Each of these classes may be individually substituted for the term "dehydration stabilizer." Accordingly, each of the following classes represents an embodiment of the present invention.

[0142] Lyoprotectants as dehydration stabilizers The dehydration stabilizer may be a lyoprotectant. A lyoprotectant is an excipient and / or additive that protects and stabilizes a biologic from damage. The term "lyoprotectant" should not be construed as being limited to the process of lyophilization. Instead, lyoprotectant should be construed in its broadest sense to include, for example, any excipient that forms hydrogen bonds with a biologic to protect the biologic from damage, as well as other mechanisms such as alteration of reaction kinetics or inhibition of migration.

[0143] Therefore, according to the present invention, a lyoprotectant is, but is not limited to, an excipient and / or additive that protects or preserves the functional activity of a biologic during either the process in which the biologic is dehydrated, such as when the biologic is converted into a dry particulate form, or after the process is completed and the biologic remains in a dry particulate form for a period of time. Those skilled in the art will readily understand that any excipient that can form hydrogen bonds with a biologic can protect the biologic from damage. Thus, in one embodiment of the present invention, whenever a dehydration stabilizer is mentioned throughout this disclosure, it can be interpreted as a lyoprotectant as defined above. Thus, according to the present invention, the dehydration stabilizer is a carbohydrate, a sugar alcohol, or a combination thereof.

[0144] Carbohydrates are a preferred class of compounds that can be used as lyoprotectants in the present invention. Carbohydrates are compounds of the general chemical formula Cx(H2O)y and are composed of molecules of carbon (C), hydrogen (H), and oxygen (O). Carbohydrates may be naturally occurring or synthetic and may be selected from monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Each of these subclasses represents a separate embodiment of the present invention. Thus, each of these carbohydrate subclasses can be used as a dehydration stabilizer for the purposes of the present invention.

[0145] Monosaccharides are simple sugars, the most common being glucose. In monosaccharides, the number of carbons usually ranges from 3 to 7. If the sugar has an aldehyde group (a functional group with the structure R-CHO), the sugar is an aldose, and if the sugar has a ketone group (a functional group with the structure RC(=O)R'), the sugar is a ketose. Depending on the number of carbons contained in the sugar, they can be trioses (3 carbons), pentoses (5 carbons), and / or hexoses (6 carbons). Galactose and fructose are other common monosaccharides. Glucose, galactose, and fructose are isomeric monosaccharides (hexoses) that have the same chemical formula but slightly different structures. Glucose and galactose are aldoses, and fructose is a ketose. Each of these examples of monosaccharides represents a separate embodiment of the present invention. Thus, each of these monosaccharides can be used as a dehydration stabilizer in the present invention.

[0146] Disaccharides are formed by the dehydration (or condensation, or dehydration synthesis) of two monosaccharides. During this process, a hydroxyl group from one monosaccharide bonds with a hydrogen from another monosaccharide, releasing a water molecule and forming a covalent bond. This is called a glycosidic bond. Glycosidic bonds (or glycosidic linkages) can be alpha or beta. An alpha bond is formed when the OH group on carbon 1 of the first glucose is below the plane of the ring, and a beta bond is formed when the OH group on carbon 1 is above the plane of the ring. The most common disaccharide is sucrose, or sugar, which is composed of glucose and fructose monomers. Another common disaccharide is trehalose, or trehalose dihydrate. Each of these disaccharide examples represents a separate embodiment of the present invention. Thus, each of these disaccharides can be used as a dehydration stabilizer in the present invention.

[0147] Oligosaccharides are carbohydrates consisting of three to six simple sugars (monosaccharides). Many oligosaccharides can be prepared by partial degradation of more complex carbohydrates (polysaccharides). Non-limiting examples of oligosaccharides include raffinose, gentianose, maltotriose, polyalditol, and cyclodextrin.

[0148] A long chain of monosaccharides linked by glycosidic bonds is a polysaccharide. The chain may be branched or unbranched and may contain various types of monosaccharides. Depending on the number of monomers attached, the molecular weight may be 100,000 daltons or more. Starch, glycogen, cellulose, and chitin are examples of polysaccharides. Dextran, chitosan, cellulose derivatives (CMC, HPC, etc.), glycosaminoglycan (HA), ficoll / polysucrose. Each of these examples of polysaccharides represents a separate embodiment of the present invention. Thus, each of these polysaccharides can be used as a dehydration stabilizer in the present invention.

[0149] Thus, according to the present invention, the dehydration stabilizer is one or more lyoprotectant(s) that can be used for the purposes of the present invention alone or in combination with other dehydration stabilizers.

[0150] Thus, the dehydration stabilizer / lyoprotectant may be one or more carbohydrate(s). Thus, the dehydration stabilizer / lyoprotectant may be a sugar. When the dehydration stabilizer / lyoprotectant is a sugar, it may be selected from the group consisting of sucrose, trehalose, raffinose, stachyose, verbascose, hydrates thereof, and combinations thereof. Preferably, it is sucrose, trehalose, trehalose dihydrate, or a combination thereof.

[0151] Thus, the dehydration stabilizer / lyoprotectant may be a sugar alcohol. When the dehydration stabilizer / lyoprotectant is a sugar alcohol, it may be selected from the group consisting of erythritol, glycerol, isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol, and combinations thereof. In some embodiments, the dehydration stabilizer is mannitol.

[0152] Thus, the dehydration stabilizer / lyoprotectant may be one or more sugar(s) and one or more sugar alcohol(s). In some embodiments, the dehydration stabilizer / lyoprotectant may be sucrose and mannitol. In some embodiments, the dehydration stabilizer / lyoprotectant may be trehalose dihydrate and mannitol.

[0153] The total concentration of lyoprotectant mixed with the biologic before the biologic is subjected to a dehydration step may be 5 mg / mL or more. Thus, the inventors have found that lower total concentrations of lyoprotectant can also be used. The inventors have found that the total concentration of lyoprotectant mixed with the biologic before the biologic is subjected to one or more dehydration steps can be 200 mg / mL to 5 mg / mL, 100 to 5 mg / mL, 85 mg / mL to 5 mg / mL, 30 mg / mL to 5 mg / mL, or about 5 mg / mL. The inventors have found that these concentrations are sufficient to protect the biologic from damage. In this context, the dehydration step can be any one of, or a combination of, freeze drying, spray drying, sterilization, and exposure to an organic solvent, such as in forming an organogel. In embodiments where multiple dehydration stabilizers are used and a lyoprotectant is one of the dehydration stabilizers used, the total concentration of the lyoprotectant may be less than 5 mg / mL, so long as the total concentration of the dehydration stabilizers mixed with the biologic before the biologic is subjected to a dehydration step is within the concentration ranges described above.

[0154] The total concentration of lyoprotectant mixed with the biologic before the biologic is exposed to the organic solvent may be 5 mg / mL or more. Thus, the inventors have discovered that a lower total concentration of lyoprotectant can be used. Thus, the total concentration of lyoprotectant mixed with the biologic before the biologic is exposed to the organic solvent may be 200-5 mg / mL, 85 mg / mL-5 mg / mL, 30 mg / mL-5 mg / mL, or about 5 mg / mL. This concentration is sufficient to protect the biologic from damage in the process of directly exposing the biologic to the organic solvent. The organic solvent may be any organic solvent that is carbon-based and liquid at room temperature and pressure. Such organic solvents may include methylene chloride, dimethyl carbonate, acetone, acetonitrile, ethyl acetate, and tetrahydrofuran. In some embodiments, the organic solvent is dimethyl carbonate. In this regard, the biologic is substantially insoluble in the organic solvent. The term "substantially insoluble" generally refers to a solubility of 0.1 mg / mL or less, such as 0.01 mg / mL or less, 0.001 mg / mL or less, etc.

[0155] In embodiments where multiple dehydration stabilizers are used and a lyoprotectant is one of the dehydration stabilizers used, the total concentration of the lyoprotectant may be less than 5 mg / mL, so long as the total concentration of the dehydration stabilizers mixed with the biologic before exposing the biologic to an organic solvent is within the concentration ranges described above.

[0156] Thus, in one embodiment, carbohydrates can be used as dehydration stabilizers at a total concentration of at least 5 mg / mL, in some embodiments, carbohydrates can be used as dehydration stabilizers at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0157] Thus, in one embodiment, sugar alcohols can be used as dehydration stabilizers at a total concentration of at least 5 mg / mL, in some embodiments, sugar alcohols can be used as dehydration stabilizers at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0158] Thus, in one embodiment, carbohydrates and sugar alcohols can be used in combination as dehydration stabilizers at a total concentration of at least 5 mg / mL. In some embodiments, carbohydrates and sugar alcohols can be used in combination as dehydration stabilizers at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 85 mg / mL, or lower concentrations of 5 mg / mL to 20 mg / mL.

[0159] Thus, in one embodiment, sugars can be used in combination as dehydration stabilizers at a total concentration of at least 5 mg / mL, in some embodiments, sugars can be used as dehydration stabilizers at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0160] Thus, in one embodiment, sucrose can be used as a dehydration stabilizer at a total concentration of at least 5 mg / mL, in some embodiments, sucrose can be used as a dehydration stabilizer at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0161] Thus, in one embodiment, trehalose dihydrate can be used as a dehydration stabilizer at a total concentration of at least 5 mg / mL, hi some embodiments, trehalose dihydrate can be used as a dehydration stabilizer at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0162] Thus, in one embodiment, a combination of trehalose dihydrate and sucrose can be used as a dehydration stabilizer at a total concentration of at least 5 mg / mL, in some embodiments, a combination of trehalose dihydrate and sucrose can be used as a dehydration stabilizer at a lower total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 85 mg / mL, or 5 mg / mL to 20 mg / mL.

[0163] Thus, in one embodiment, a combination of trehalose dihydrate and mannitol, or a combination of sucrose and mannitol, can be used as a dehydration stabilizer at a total concentration of at least 5 mg / mL. In some embodiments, a combination of trehalose dihydrate and mannitol, or a combination of sucrose and mannitol, can be used as a dehydration stabilizer at a lower concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 85 mg / mL, or 5 mg / mL to 20 mg / mL.

[0164] Synthetic polymers as dehydration stabilizers According to the present invention, synthetic polymers may also be used as dehydration stabilizers.

[0165] One or more synthetic polymers from the group containing one or more polyalkylene glycol units can be used, such as polyethylene glycol (PEG), polypropylene glycol, poly(ethylene glycol)-block-poly(propylene glycol) copolymers, or polyethylene oxide, polypropylene oxide, polyvinyl alcohol, poly(vinylpyrrolidinone), polylactic acid, polylactic acid-co-glycolic acid, random or block copolymers, or any combination / mixtures thereof, although this list is not intended to be limiting.

[0166] Polymers used as dehydration stabilizers can be branched (multi-armed) or linear. In branched polymers, the core refers to a continuous portion of the molecule joined to arms extending from the core, the arms often having functional groups at the ends of the branches. It is noted that these polymers may have, for example, 2-100 arms, each arm having a terminus, and some precursors may be dendrimers or other highly branched materials. An arm refers to a linear chain of chemical groups connecting the crosslinkable functional groups to the polymer core. In some embodiments, polymers that can be used as dehydration stabilizers can include 3-300 arms. One of ordinary skill in the art will readily appreciate that all ranges and values ​​within the explicitly stated ranges are contemplated, for example, 4, 6, 8, 10, 12, 4-16, 8-100, 6, 8, 10, 12, or at least 4 arms.

[0167] The polymers used as dehydration stabilizers may or may not be "functional polymers". "Functional polymers" and "non-functional polymers" have been defined previously in this disclosure. Functional groups are generally configured to have reactive groups for polymerization, or to react with each other in an electrophile-nucleophile reaction, or to participate in other polymerization reactions. In some embodiments, the polymers used as dehydration stabilizers include a nucleophile or an electrophile.

[0168] Thus, according to the present invention, the dehydration stabilizer is one or more polymer(s) that can be used for the purposes of the present invention alone or in combination with other dehydration stabilizers. In a preferred embodiment, the polymer is poly(ethylene) oxide, commonly known as polyethylene glycol.

[0169] In certain embodiments, when the dehydration stabilizer is polyethylene glycol, its average molecular weight may be in the range of about 2,000 to about 100,000 daltons, or in the range of about 10,000 to about 60,000 daltons, or in the range of about 15,000 to about 50,000 daltons. In certain embodiments, the average molecular weight may be in the range of about 10,000 to about 40,000 daltons, or about 20,000 daltons. The average molecular weight is given as the number average molecular weight (Mn), which in certain embodiments may be determined by MALDI.

[0170] The total concentration of polymer mixed with the biologic before the biologic is subjected to a dehydration step may be 5 mg / mL or more. Thus, the inventors have found that lower total concentrations of polymer can also be used. The inventors have found that the total concentration of polymer mixed with the biologic before the biologic is subjected to one or more dehydration steps can be 200 mg / mL to 5 mg / mL, 100 to 5 mg / mL, 55 mg / mL to 5 mg / mL, 30 mg / mL to 5 mg / mL, or about 5 mg / mL. The inventors have found that these concentrations are sufficient to protect the biologic from damage. In this context, the dehydration step can be any one of, or a combination of, freeze drying, spray drying, sterilization, and exposure to an organic solvent, such as in forming an organogel. In embodiments where multiple dehydration stabilizers are used and a polymer is one of the dehydration stabilizers used, the total concentration of polymer can be as low as 5-10 mg / mL, so long as the total concentration of dehydration stabilizers mixed with the biologic before the biologic is subjected to a dehydration step is within the concentration ranges described above.

[0171] The total concentration of the polymer mixed with the biologic before the biologic is exposed to the organic solvent may be 5 mg / mL or more. Thus, the inventors have discovered that a lower total concentration of the polymer can be used. Thus, the total concentration of the polymer mixed with the biologic before the biologic is exposed to the organic solvent can be 200 mg / mL to 5 mg / mL, 100 to 5 mg / mL, 55 mg / mL to 5 mg / mL, 30 mg / mL to 5 mg / mL, or about 5 mg / mL. This concentration is sufficient to protect the biologic from damage in the process of directly exposing the biologic to the organic solvent. The organic solvent can be any organic solvent that is carbon-based and liquid at room temperature and pressure. Such organic solvents can include methylene chloride, dimethyl carbonate, acetone, acetonitrile, ethyl acetate, and tetrahydrofuran. In some embodiments, the organic solvent is dimethyl carbonate. In this context, the biologic is substantially insoluble in the organic solvent. The term "substantially insoluble" generally refers to a solubility of 0.1 mg / mL or less, e.g., 0.01 mg / mL or less, 0.001 mg / mL or less, etc. In embodiments where multiple dehydration stabilizers are used and a lyoprotectant is one of the dehydration stabilizers used, the total concentration of the lyoprotectant may be less than 5 mg / mL, so long as the total concentration of the dehydration stabilizers mixed with the biologic prior to exposing the biologic to an organic solvent is within the concentration ranges described above.

[0172] Thus, in one embodiment, the synthetic polymer can be used as a dehydration stabilizer at a total concentration of at least 5 mg / mL, hi some embodiments, the synthetic polymer can be used as a dehydration stabilizer at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0173] Thus, in one embodiment, the PEG polymer can be used as a dehydration stabilizer at a total concentration of at least 5 mg / mL, hi some embodiments, the PEG polymer can be used as a dehydration stabilizer at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0174] Thus, in one embodiment, the multi-arm PEG polymer can be used as a dehydration stabilizer at a total concentration of at least 5 mg / mL, hi some embodiments, the multi-arm PEG polymer can be used as a dehydration stabilizer at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0175] Thus, in one embodiment, multi-arm PEG polymers containing nucleophilic or electrophilic groups can be used as dehydration stabilizers at a total concentration of at least 5 mg / mL, hi some embodiments, multi-arm PEG polymers containing nucleophilic or electrophilic groups can be used as dehydration stabilizers at a total concentration of 5 mg / mL to 200 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 85 mg / mL, or as low as 5 mg / mL to 20 mg / mL.

[0176] Thus, in one embodiment, multi-arm PEG polymers containing nucleophilic or electrophilic groups can be used as dehydration stabilizers at a total concentration of at least 5 mg / mL. In some embodiments, multi-arm PEG polymers containing nucleophilic or electrophilic groups can be used as dehydration stabilizers at a total concentration as low as 5 mg / mL to 200 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 85 mg / mL, or 5 mg / mL to 20 mg / mL. In some embodiments, the nucleophile is an amine, such as a primary amine.

[0177] Dehydration stabilizer combination Those skilled in the art will understand that any of the above dehydration stabilizers can be used in any dehydration step at any concentration range. Thus, for example, in one embodiment, a combination of a carbohydrate, such as sucrose or trehalose dihydrate, and a PEG polymer, such as a multi-arm PEG polymer containing an amine, can be used as a dehydration stabilizer. In this embodiment, the total concentration of all dehydration stabilizers can be as low as 5 mg / mL to 200 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 85 mg / mL, or 5 mg / mL to 20 mg / mL.

[0178] Other stabilizers According to the present invention, other stabilizers can also be used in combination with the above-mentioned dehydration stabilizers, and can be selected from buffers, salts, amino acids, surfactants, and antioxidants, in known concentration ranges within the knowledge of those skilled in the art. In some embodiments, at least one dehydration stabilizer can be used with at least one or at least two stabilizers. For example, at least one dehydration stabilizer is used in combination with a buffer, such as PBS, or in combination with a surfactant, such as a non-ionic surfactant, or both.

[0179] The term "nonionic surfactant" refers to a surfactant that does not contain positively or negatively charged functional groups. In contrast to anionic and cationic surfactants, nonionic surfactants do not ionize in solution. The nonionic surfactant may be a poloxamer. Poloxamers are nonionic triblock copolymers consisting of a central hydrophobic polychain (propylene oxide) flanked by hydrophilic polychains (ethylene oxide). The length of the polymer blocks can be customized, resulting in a variety of poloxamers with slightly different properties. Thus, the nonionic surfactant may be Pluronic F127 (Poloxamer 407), Pluronic F123 (Poloxamer 403), Pluronic F-68 (Poloxamer 188), Pluronic P123, Pluronic P85, or other polyethylene oxide-polypropylene oxide (EO-PO) block copolymers with over 3,000-4,000 MW, or combinations thereof.

[0180] Thus, in one embodiment, at least one dehydration stabilizer can be used in any concentration as described above, along with at least one or at least two stabilizers, e.g., a buffer containing at least two or at least three salts, and a non-ionic surfactant, such as F-68, F-127, or F123.

[0181] Particles in the implant of the present invention In some embodiments, a particle or all particles in an implant of the invention, such as a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic as described above and at least one dehydration stabilizer as described above.

[0182] In some embodiments, an implant of the invention, e.g., a particle or the whole particle in a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0183] In some embodiments, an implant of the invention, e.g., a particle in a pharma- ceutically acceptable implant of the invention, or the entire particle, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, e.g., F-68, F-127, or F123.

[0184] In some embodiments, a particle, or the entire particle, in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0185] In some embodiments, the particles, or the entire particles, in the implants of the invention, e.g., the pharma- ceutically acceptable implants of the invention, are comprised of a mixture of the biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, e.g., F-68, F-127, or F123.

[0186] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, for example, a pharma- ceutically acceptable implant of the invention that includes a mixture of a biologic as described above and at least one dehydration stabilizer as described above.

[0187] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention that includes a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0188] In some embodiments, Dv 90Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, comprising a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a mixture of non-ionic surfactants, such as F-68, F-127, or F123.

[0189] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention that comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0190] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, which comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, such as F-68, F-127, or F123.

[0191] Method for protecting biological products in processes in which the biological products are directly exposed to organic solvents The present invention also provides a method for protecting a biological product from damage during a process in which the biological product is directly exposed to an organic solvent. The method includes mixing the biological product with at least one dehydration stabilizer prior to directly exposing the biological product to an organic solvent. The at least one dehydration stabilizer is described in detail in the previous section.

[0192] In one embodiment, the dehydration stabilizer and biologic are substantially insoluble in organic solvents, e.g., solubility of 0.1 mg / mL or less, such as 0.01 mg / mL or less, such as 0.001 mg / mL or less.

[0193] In some embodiments, before the biologic is exposed to an organic solvent, the biologic is mixed with at least one dehydration stabilizer that is a carbohydrate, a sugar alcohol, or a combination thereof. In another preferred embodiment, the at least one dehydration stabilizer is selected from a sugar, a sugar alcohol, or a combination thereof. Such sugars may include sucrose, trehalose, raffinose, stachyose, verbascose, hydrates thereof, and combinations thereof, such as sucrose, trehalose, trehalose dihydrate, and combinations thereof. Such sugar alcohols may include erythritol, glycerol, isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol, and combinations thereof. In this embodiment, the mixture may include additional stabilizers, such as, for example, a buffer and a non-ionic surfactant.

[0194] In another embodiment, before the biologic is exposed to an organic solvent, the biologic is mixed with at least one dehydration stabilizer that is a synthetic polymer. For this purpose, any synthetic polymer defined in the previous section can be used. Such synthetic polymers can include polyalkylene oxides such as polyethylene glycol, polyvinylpyrrolidinone, and polyvinyl alcohol. In this embodiment, the mixture can include additional stabilizers, such as, for example, a buffer and a non-ionic surfactant.

[0195] In another embodiment, before the biologic is exposed to an organic solvent, the biologic is mixed with at least two dehydration stabilizers selected from carbohydrates and synthetic polymers, as described above and in the previous section, In this embodiment, the mixture may include additional stabilizers, such as, for example, a buffer and a non-ionic surfactant.

[0196] In such embodiments, the biologic is a recombinant protein, a lipid encapsulating a nucleic acid, or a virus that contains at least one heterologous nucleic acid sequence. In some embodiments, the virus is selected from the group consisting of AAV1, AAV2, such as AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In these embodiments, the AAV contains at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can code for a therapeutic protein, as described in the section under the heading of biologics.

[0197] In another preferred embodiment, the recombinant protein is selected from the group consisting of an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0198] In one embodiment, the biologic is a virus, and the total amount of the virus mixed with the at least one dehydration stabilizer is at least 10 9 In certain embodiments, the total amount of virus mixed with at least one dehydration stabilizer is 10 9 ~10 15 In another preferred embodiment, the total amount of virus mixed with at least one dehydration stabilizer is 10 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 vg.

[0199] In one embodiment, the biologic to be protected from damage is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In one embodiment, the total amount of AAV mixed with at least one dehydration stabilizer is at least 10 9 In certain embodiments, the total amount of AAV mixed with at least one dehydration stabilizer is 10 9 ~10 15 In another preferred embodiment, the total amount of AAV mixed with at least one dehydration stabilizer is 10 vg. 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 vg.

[0200] In one embodiment, the biologic to be protected from damage is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In one embodiment, the total amount of AAV mixed with at least one dehydration stabilizer is at least 10 9 In certain embodiments, the total amount of AAV mixed with at least one dehydration stabilizer is 10 10 ~10 15 In another preferred embodiment, the total amount of AAV mixed with at least one dehydration stabilizer is 10 vg. 10 ~10 15 vg, or 10 10 ~10 14 vg, or 10 10 ~10 13 vg, or 10 10 ~10 12 vg, or 10 10 ~10 11 vg.

[0201] In one embodiment, the biologic is a recombinant protein, and the total amount of recombinant protein mixed with at least one dehydration stabilizer is at least 10 μg. In a particular embodiment, the total amount of recombinant protein mixed with at least one dehydration stabilizer is 10-3000 μg. In other preferred embodiments, the total amount of recombinant protein mixed with at least one dehydration stabilizer is 10-2,500 μg, or 10-2000 μg.

[0202] In one embodiment, the invention disclosed herein does not relate to adsorbing a biologic to a silica particle, such as a mesoporous silica particle, or equivalent, which means that the biologic is not protected by the mesoporous silica particle at any point during the manufacturing process of an implant, such as a pharma- ceutically acceptable implant of the present invention.

[0203] In one embodiment, the invention disclosed herein does not relate to adsorbing a biologic to a fatty acid moiety or equivalent, meaning that at no point during the manufacturing process of an implant, such as a pharma- ceutically acceptable implant of the present invention, the biologic is not protected by a fatty acid moiety.

[0204] In one embodiment, the invention disclosed herein does not relate to adsorbing AAV to silica particles, such as mesoporous silica particles, or the like, which means that the AAV is not protected by mesoporous silica particles at any point during the manufacturing process of an implant, such as a pharma- ceutically acceptable implant of the invention.

[0205] In one embodiment, the invention disclosed herein does not relate to adsorbing AAV to a fatty acid moiety or equivalent, meaning that at no point during the process of manufacturing an implant, such as a pharma- ceutically acceptable implant of the present invention, the AAV is not protected by a fatty acid moiety.

[0206] Method for producing a pharma-ceutically acceptable implant According to the present invention, there is provided a method of producing a pharma- ceutically acceptable implant comprising a biologic, comprising forming an organogel comprising the biologic, comprising forming a matrix comprising at least two covalently crosslinked multi-arm precursors in an organic solvent in the presence of the biologic, followed by forming a xerogel, comprising removing the organic solvent.

[0207] (a) providing a mixture of a biological agent and at least one dehydration stabilizer; The method of producing a pharma- ceutically acceptable implant first requires providing a mixture of a biologic and at least one dehydration stabilizer.

[0208] In some embodiments, the biologic is a recombinant protein, a lipid encapsulating a nucleic acid, or a virus comprising a heterologous nucleic acid sequence. In some embodiments, the virus is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can code for a therapeutic protein, as described in the section under the heading Biologics. In certain embodiments, the AAV is administered in a pharmaceutically acceptable implant at least 10 13 vg / cm 3 In some embodiments, the AAV may be present in the pharma- ceutically acceptable implant at a total concentration of at least 10 14 In one embodiment, the total amount of AAV is 10 9 ~10 15In some embodiments, the total amount of AAV in the pharma- ceutical implant is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV in the pharma- ceutical implant is on the order of 10 10 ~10 15 vg, or 10 10 ~10 14 vg, or 10 10 ~10 13 vg, or 10 10 ~10 12 vg, or 10 10 ~10 11 It is on the order of vg.

[0209] In another preferred embodiment, the recombinant protein is selected from the group consisting of an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone. In one embodiment, the total amount of recombinant protein in the pharmaceutically acceptable implant is at least 10 μg. In a particular embodiment, the total amount of recombinant protein in the pharmaceutically acceptable implant is 10-3000 μg. In other preferred embodiments, the total amount of recombinant protein in the pharmaceutically acceptable implant is 5-2500 μg, or 5-2000 μg.

[0210] In some embodiments, at least one dehydration stabilizer is a carbohydrate, a sugar alcohol, and a combination thereof. In another embodiment, at least one dehydration stabilizer is a synthetic polymer. In another embodiment, at least one dehydration stabilizer is two dehydration stabilizers selected from carbohydrates and synthetic polymers. The dehydration stabilizer can be selected from sugars, sugar alcohols, or combinations thereof. Such sugars can include sucrose, trehalose, raffinose, stachyose, verbascose, hydrates thereof, and combinations thereof, such as sucrose, trehalose, trehalose dihydrate, and combinations thereof. Such sugar alcohols can include erythritol, glycerol, isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol, and combinations thereof. When the dehydration stabilizer is a synthetic polymer, the dehydration stabilizer can be a polyalkylene oxide, such as polyethylene glycol, polyvinylpyrrolidinone, and polyvinyl alcohol. The total concentration of the dehydration stabilizer mixed with the total biologic amount is at least 5 mg / mL, such as 5 mg / mL to 200 mg / mL, 5 mg / mL to 100 mg / mL, 5 mg / mL to 85 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 30 mg / mL, or 5 mg / mL to 15 mg / mL.

[0211] At this step, in some embodiments, the mixture may also contain other stabilizing agents, such as buffers and non-ionic surfactants.

[0212] (b) providing at least one multi-arm precursor; In one embodiment, at least one multi-arm precursor is provided. The precursors and multi-arm precursors used in the present invention are described in detail in the section entitled "Xerogels". In some embodiments, the at least one multi-arm precursor comprises at least eight arms, or at least four arms. The at least one multi-arm precursor comprises an electrophile or a nucleophile.

[0213] In another embodiment, the at least one multi-arm precursor comprises at least two multi-arm precursors. In such an embodiment, one multi-arm precursor comprises an electrophile and another multi-arm precursor comprises a nucleophile. In another embodiment, the at least one multi-arm precursor comprises at least two multi-arm precursors, each of which comprises an electrophile.

[0214] In these embodiments, the nucleophile may be an amine, such as a primary amine, a thiol, an azide, or a hydrazide, and the electrophile may be a succinimidyl ester, succinimidyl carbonate, nitrophenyl carbonate, an aldehyde, a ketone, an acrylate, an acrylamide, a maleimide, a vinyl sulfone, an iodoacetamide, an alkene, an alkyne, a dibenzocyclooctyne, a norbornene, an epoxide, a mesylate, a tosylate, a tresyl, a cyanurate, an orthopyridyl disulfide, or a halide. In one embodiment of the invention, when the electrophile is a succinimidyl ester, the electrophile may include a reactive group such as succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), or succinimidyl glutaramide.

[0215] In some embodiments, at least one multi-arm precursor is a first multi-arm PEG precursor that comprises a primary amine.

[0216] (c) providing at least one further multi-arm precursor; In one embodiment, at least one additional multi-arm precursor is provided. The precursors and multi-arm precursors used in the present invention are described in detail in the section entitled "Xerogels". In some embodiments, the at least one additional multi-arm precursor comprises at least eight arms, or at least four arms. The at least one additional multi-arm precursor may comprise an electrophile or a nucleophile.

[0217] In another embodiment, the at least one additional multi-arm precursor comprises at least two multi-arm precursors. In such an embodiment, one multi-arm precursor comprises an electrophile and another multi-arm precursor comprises a nucleophile. In another embodiment, the at least one additional multi-arm precursor comprises at least two multi-arm precursors, each of which comprises an electrophile.

[0218] In these embodiments, the nucleophile can be an amine, such as a primary amine, a thiol, an azide or a hydrazide, and the electrophile can be a succinimidyl ester, succinimidyl carbonate, nitrophenyl carbonate, an aldehyde, a ketone, an acrylate, an acrylamide, a maleimide, a vinyl sulfone, an iodoacetamide, an alkene, an alkyne, a dibenzocyclooctyne, a norbornene, an epoxide, a mesylate, a tosylate, a tresyl, a cyanurate, an orthopyridyl disulfide, or a halide.

[0219] In some embodiments, the at least one additional multi-arm precursor comprises at least two additional multi-arm precursors, including a first multi-arm precursor comprising an electrophile comprising a first reactive group and a second multi-arm precursor comprising an electrophile comprising a second reactive group. In one embodiment of the present invention, when the electrophile is a succinimidyl ester, the first reactive group and the second reactive group are selected from succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), or succinimidyl glutaramide.

[0220] Processing Steps Next, (a), (b), and (c) above are processed, respectively, to obtain (d), (c), and (f).

[0221] (d) Particles comprising a mixture of a biological agent and at least one dehydration stabilizer. Processing (a) to obtain (d) may include one or more dehydration steps, including forming dry particulates of a mixture of the biologic and at least one dehydration stabilizer. Such methods are known in the art and include, but are not limited to, freeze drying, spray drying, or vacuum drying. In one embodiment, the mixture of the biologic and at least one dehydration stabilizer is converted to a dry particulate form. In accordance with the present invention, the dehydration stabilizer protects the biologic from damage during one or more dehydration steps.

[0222] (c) and (f) - Processed (b) and (c) In one embodiment, processing (b) and (c) may or may not include one or more dehydration steps, such as freeze drying, spray drying, or vacuum drying, to convert the multi-arm precursor to a dry particulate form. In one embodiment, if these dehydration steps are not employed, a sterilization step, such as gamma radiation sterilization, electron beam sterilization, or ethylene oxide sterilization, may be employed.

[0223] (g) and (h) - Adding an organic solvent to (c) and (f) In one embodiment, an organic solvent may be added to each of (c) and (f) to obtain (g) and (h). In this embodiment, the organic solvent may be any organic solvent that is carbon-based and liquid at room temperature and pressure. Such organic solvents may include methylene chloride, dimethyl carbonate, acetone, acetonitrile, ethyl acetate, and tetrahydrofuran. In some embodiments, the organic solvent is dimethyl carbonate.

[0224] Organogel formation In one embodiment, (g) is mixed with (d) to obtain (i), which is then mixed with (h). In another embodiment, (h) is mixed with (d) to obtain (i), which is then mixed with (g). In this step, the biologic is directly exposed to the organic solvent. According to the present invention, the biologic is in the form of particles comprising a mixture of the biologic and at least one dehydration stabilizer, and thus the biologic is protected from damage by the at least one dehydration stabilizer. In this step, the organic solvent and the dehydration stabilizer are substantially insoluble in the organic solvent, e.g., solubility of 0.1 mg / mL or less, such as 0.01 mg / mL or less, 0.001 mg / mL or less.

[0225] In this step, at least two multi-arm precursors react in an electrophile-nucleophile reaction to form a covalently crosslinked matrix, i.e., an organogel. In some embodiments, at least three multi-arm precursors react in an electrophile-nucleophile reaction to form a covalently crosslinked matrix, i.e., an organogel.

[0226] Xerogel formation According to the present invention, forming a xerogel from an organogel includes a drying step. Those skilled in the art will appreciate that all known drying methods can be used. Possible processes include, for example, precipitation with a non-solvent, nitrogen sweep drying, vacuum drying, freeze drying, a combination of heat and vacuum, and lyophilization. In some embodiments, the organogel is dried in a nitrogen gas stream at a temperature of 35° C. to 37° C. for 1 to 5 days, or at least 4 days, or at least 3 days. In some embodiments, prior to the drying step, the organogel is placed in a tube of predetermined dimensions and formed into a particular shape. In some embodiments, the pharma-ceutically acceptable implant is in the form of a fiber, the fiber being characterized by a diameter of about 0.1 mm or more and / or a length of about 2.0 mm or more.

[0227] In one embodiment, (b) is part of (a), and thus (b) is a dehydration stabilizer for the biologic. In this embodiment, (d) comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer, said dehydration stabilizer being (b).

[0228] In one embodiment, (b) is part of (a), and thus (b) is a dehydration stabilizer for the biologic. In this embodiment, (d) comprises a particle comprising a mixture of the biologic and at least one dehydration stabilizer. The at least one dehydration stabilizer may be at least two dehydration stabilizers, including (b) as a first dehydration stabilizer and another dehydration stabilizer, such as a lyoprotectant, such as a carbohydrate, such as a sugar (e.g., sucrose and trehalose), or a sugar alcohol (e.g., mannitol).

[0229] In certain embodiments, (b) or (c) further comprises a polymer, such as a synthetic polymer, that is a "non-functional polymer." A "non-functional" polymer as described above in this disclosure is a polymer that does not participate in a cross-linking reaction between multi-arm precursors.

[0230] The polymer may be further defined as a polymer that dissolves in both organic solvents and water. In one embodiment, the additional polymer is a non-functional polymer used as a bulking agent in pharma-ceutically acceptable implants. The MW of the polymer may be 1,000-35,000 Da, for example, 5,000-35,000 Da, for example, 5,000-10,000 Da, 7,000-10,000 Da, 8,000-15,000 Da, 8,000-25,000 Da, or 5,000 Da or more.

[0231] The additional polymers may be branched (multi-armed) or linear. In the case of branched polymers, the core refers to a continuous portion of the molecule joined to arms extending from the core, the arms often having functional groups at the ends of the branches. It is noted that these polymers may have, for example, 2-100 arms, each arm having a terminus, and some precursors may be dendrimers or other highly branched materials. The arms refer to linear chains of chemical groups connecting the crosslinkable functional groups to the polymer core. In some embodiments, these additional polymers may include 3-300 arms. One of ordinary skill in the art will immediately appreciate that all ranges and values ​​within the explicitly stated ranges are contemplated, for example, 4, 6, 8, 10, 12, 4-16, 8-100, 6, 8, 10, 12, or at least 4 arms.

[0232] In various embodiments, the additional polymer is selected from the group consisting of polyalkylene oxides, such as polyethylene glycol, polyvinylpyrrolidinone, and polyvinyl alcohol.

[0233] In one embodiment, the invention disclosed herein does not relate to adsorbing a biologic to silica particles, such as mesoporous silica particles, or the like, at any point during the process for manufacturing an implant, such as a pharma- ceutically acceptable implant, of the present invention.

[0234] In one embodiment, the invention disclosed herein does not relate to adsorbing a biologic to a fatty acid moiety or equivalent at any point during the process for manufacturing an implant, such as a pharma- ceutically acceptable implant of the present invention.

[0235] In one embodiment, the invention disclosed herein does not relate to adsorbing AAV to silica particles, such as mesoporous silica particles, or the like, at any point during the process for producing an implant, such as a pharma- ceutically acceptable implant, of the invention.

[0236] In one embodiment, the invention disclosed herein does not relate to adsorbing AAV to a fatty acid moiety or equivalent at any point during the process for producing an implant, such as a pharma- ceutically acceptable implant of the invention.

[0237] Method for producing pharma-ceutically acceptable implants for the controlled release of biologics - Patent Application 20070123333 According to the present invention, there is provided a method for preparing a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic. The method is essentially the same as in the previous section. Steps (a), (b), and (c) of the previous section may require further consideration.

[0238] Throughout this section, the (w / w) percentages are based on the weight of the pharma- ceutically acceptable implant.

[0239] Throughout this section, in all embodiments, including all parameters disclosed in this section, the terms "particle" or "total particles" or "Dv 90 Each of the "particle sizes" is described as relating to particles that include a mixture of a biologic and at least one dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof.

[0240] Throughout this section, in all embodiments, including all parameters disclosed in this section, the term "particle" or "total particles" or "Dv 90 "Particle size" may also refer to the mixture described in the following paragraph.

[0241] In some embodiments, a particle or all particles in an implant of the invention, such as a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic as described above and at least one dehydration stabilizer as described above.

[0242] In some embodiments, an implant of the invention, e.g., a particle or the whole particle in a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0243] In some embodiments, an implant of the invention, e.g., a particle in a pharma- ceutically acceptable implant of the invention, or the entire particle, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, e.g., F-68, F-127, or F123.

[0244] In some embodiments, a particle, or the entire particle, in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0245] In some embodiments, the particles, or the entire particles, in the implants of the invention, e.g., the pharma- ceutically acceptable implants of the invention, are comprised of a mixture of the biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, e.g., F-68, F-127, or F123.

[0246] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, for example, a pharma- ceutically acceptable implant of the invention that includes a mixture of a biologic as described above and at least one dehydration stabilizer as described above.

[0247] In some embodiments, Dv 90Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention that includes a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0248] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, comprising a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a mixture of non-ionic surfactants, such as F-68, F-127, or F123.

[0249] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention that comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0250] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, which comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, such as F-68, F-127, or F123.

[0251] Some aspects of the present disclosure are directed to a method of making a pharma- ceutical acceptable implant for controlled release of a biologic, comprising forming a xerogel comprising at least two covalently crosslinked precursors in which particles comprising a biologic and at least one dehydration stabilizer are dispersed. In one embodiment, the at least one dehydration stabilizer is selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The inventors have discovered that the total (w / w) % of the dehydration stabilizer in the pharma- ceutical acceptable implant can be selected to be 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less to allow for controlled release of the biologic. In a related or separate embodiment, the molecular weight between crosslinks of the xerogel can be selected from 7-25 kDa, 9-20 kDa, or 10-15 kDa to allow for controlled release of the biologic. In another related or separate embodiment, the % (w / w) of the total particles comprising the mixture of biologic and said dehydration stabilizer can be selected to be 80% or less, or 70% or less, or 60% or less, or 50% or less, e.g., 20%-40%, 20%-30%, etc., to allow for controlled release of the biologic. In another related or separate embodiment, the % (w / w) of the total number of multi-arm precursors can be selected from 20%-80%, e.g., 35%-75%, e.g., 35%-65%, 35%-55%, 35%-45%, etc., to allow for controlled release of the biologic. In another related or separate embodiment, the ratio of (1) the % (w / w) of total particles comprising the mixture of dehydration stabilizers to (2) the % (w / w) of the total number of multi-arm precursors can be selected from 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0, to enable controlled release of the biologic.In another related or separate embodiment, Dv. 90 The particle size may be selected to be between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, to allow for controlled release of the biologic, and the particles include a mixture of the biologic and said dehydration stabilizer, such as a carbohydrate, sugar alcohol, or combinations thereof. These individual parameters may be selected either alone or in combination with each other to allow for controlled release of the biologic.

[0252] A method of providing a pharma- ceutically acceptable implant for controlled release of a biologic comprises forming a xerogel comprising at least three multi-arm precursors, the at least three multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group, the at least three multi-arm precursors having dispersed therein particles comprising the biologic and at least one dehydration stabilizer. In one embodiment, the hydrolysis half-life of the third multi-arm precursor is longer than that of the second precursor. The inventors have discovered that the molar ratio of the first reactive group in the second multi-arm precursor to the second reactive group in the third multi-arm precursor can be selected from 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40 to enable controlled release of the biologic. In some embodiments, the nucleophile is a primary amine and the electrophile is a succinimidyl ester. In some embodiments, the first reactive group is succinimidyl succinate and the second reactive group is succinimidyl glutarate. In one embodiment, the at least one dehydration stabilizer is selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The inventors have discovered that the total (w / w) % of the dehydration stabilizer in the pharma- ceutical acceptable implant can be selected to be 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less to allow for controlled release of the biologic. In a related or separate embodiment, the molecular weight between crosslinks of the xerogel can be selected from 7-25 kDa, 9-20 kDa, or 10-15 kDa to allow for controlled release of the biologic. In another related or separate embodiment, the (w / w)% of the total particle comprising the mixture of biologic and said dehydration stabilizer may be selected to be 50% or less, e.g., 20%-40%, 20%-30%, etc., to allow for controlled release of the biologic.In another related or separate embodiment, the % (w / w) of the total number of multi-arm precursors may be selected from 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc. to allow for controlled release of the biologic. In another related or separate embodiment, the ratio of (1) the % (w / w) of the total particles comprising the mixture of dehydration stabilizers to (2) the % (w / w) of the total number of multi-arm precursors may be selected from 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0, to enable controlled release of the biologic. In another related or separate embodiment, Dv. 90 The particle size may be selected to be between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, to allow for controlled release of the biologic, and the particles include a mixture of the biologic and said dehydration stabilizer, such as a carbohydrate, sugar alcohol, or combinations thereof. These individual parameters may be selected either alone or in combination with each other to allow for controlled release of the biologic.

[0253] Some aspects of the present disclosure are directed to a method of manufacturing a pharma- ceutically acceptable implant for controlled release of a biologic, comprising forming an organogel comprising at least two covalently crosslinked precursors in which particles comprising a biologic and at least one dehydration stabilizer are dispersed. In one embodiment, the organogel is formed by adding an organic solvent to each multi-arm precursor and mixing them. In one embodiment, the total (w / v) % of the multi-arm precursor dissolved in the organic solvent can be selected to allow for controlled release of the biologic. In one embodiment, the at least one dehydration stabilizer is selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The inventors have discovered that the total (w / w) % of the dehydration stabilizer in the pharma-ceutically acceptable implant can be selected to allow for controlled release of the biologic, to be 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. In a related or separate embodiment, the molecular weight between crosslinks of the xerogel may be selected from 7-25 kDa, 9-20 kDa, or 10-15 kDa to allow for controlled release of the biologic. In another related or separate embodiment, the (w / w)% of the total particles comprising the mixture of the biologic and the dehydration stabilizer may be selected to be 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc., to allow for controlled release of the biologic. In another related or separate embodiment, the (w / w)% of the total number of multi-arm precursors may be selected from 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc., to allow for controlled release of the biologic.In another related or separate embodiment, the ratio of (1) the % (w / w) of total particles comprising the mixture of dehydration stabilizers to (2) the % (w / w) of the total number of multi-arm precursors can be selected from 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0, to enable controlled release of the biologic. In one embodiment, the organogel and xerogel comprise at least three multi-arm precursors, including a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group. In one embodiment, the hydrolysis half-life of the third multi-arm precursor is longer than that of the second precursor. The inventors have discovered that the molar ratio of the first reactive group in the second multi-arm precursor to the second reactive group in the third multi-arm precursor can be selected from 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40 to enable controlled release of the biologic. In some embodiments, the nucleophile is a primary amine and the electrophile is a succinimidyl ester. In some embodiments, the first reactive group is succinimidyl succinate and the second reactive group is succinimidyl glutarate. In another related or separate embodiment, Dv. 90 The particle size may be selected to be between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, to allow for controlled release of the biologic, and the particles include a mixture of the biologic and said dehydration stabilizer, such as a carbohydrate, sugar alcohol, or combinations thereof. These individual parameters may be selected either alone or in combination with each other to allow for controlled release of the biologic.

[0254] In one embodiment, the controlled release of the biologic is characterized by the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 2 days. In another embodiment, the amount of biologic released on day 1 is 0-25%, 0-20%, 0-10%, 0-5%, or about 0% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 3 days but not more than 30 days, not more than 25 days, or not more than 16 days.

[0255] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic, which number of days is at least 2 days, at least 3 days, or at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less.

[0256] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 2 days, at least 3 days, or at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 60% or less.

[0257] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic, which number of days is at least 2 days, at least 3 days, or at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 55% or less.

[0258] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic, which number of days is at least 2 days, at least 3 days, or at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 50% or less.

[0259] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic, which number of days is at least 2 days, at least 3 days, or at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 45% or less.

[0260] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic, which number of days is at least 2 days, at least 3 days, or at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 40% or less.

[0261] In one embodiment, where the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 3 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 60% or less.

[0262] In one embodiment, where the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 3 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 55% or less.

[0263] In one embodiment, where the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 3 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 50% or less.

[0264] In one embodiment, where the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 3 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 45% or less.

[0265] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 3 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 40% or less.

[0266] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 60% or less.

[0267] In one embodiment, where the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 55% or less.

[0268] In one embodiment, when the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 50% or less.

[0269] In one embodiment, where the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 45% or less.

[0270] In one embodiment, where the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic being at least 4 days, the total (w / w) % of carbohydrate, sugar alcohol, or combination thereof is selected to be 40% or less.

[0271] In one embodiment, when the controlled release of the biologic is characterized in that the amount of biologic released on day 1 is 0-50% of the total amount of the biologic, Dv 90 The particle size can be selected to be between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of a biologic and at least one dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. 90 The larger the particle size, the higher the percentage of the total amount of biologic released on day 1.

[0272] In some embodiments, when the amount of biologic released on day 1 is 0-50%, 0-45%, 0-40%, 0-35%, 0-30%, 0-25%, 0-20%, 0-15%, 0-10%, 0-5% of the total amount of biologic, Dv 90 The particle size may be selected to be 10-20 μm, 10-30 μm, 10-40 μm, 10-50 μm, 10-60 μm, 10-70 μm, 10-100 μm, 10-110 μm, 10-120 μm, 10-130 μm, 10-140 μm, 10-150 μm, 10-160 μm, 10-170 μm, 10-180 μm, 10-190 μm, 10-200 μm, and the particles include a mixture of a biologic and at least one dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. 90 The larger the particle size, the higher the percentage of the total amount of biologic released on day 1.

[0273] In one embodiment, the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic, which is at least 2 days, at least 3 days, or at least 4 days, and the Dv 90The particle size can be selected to be between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of a biologic and at least one dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. 90 The larger the particle size, the higher the percentage of the total amount of biologic released on day 1.

[0274] In some embodiments, the controlled release of the biologic is characterized by the number of days required for 100% release of the biologic, where the number of days is at least 2 days, at least 3 days, or at least 4 days. 90 The particle size may be selected to be 10-20 μm, 10-30 μm, 10-40 μm, 10-50 μm, 10-60 μm, 10-70 μm, 10-100 μm, 10-110 μm, 10-120 μm, 10-130 μm, 10-140 μm, 10-150 μm, 10-160 μm, 10-170 μm, 10-180 μm, 10-190 μm, 10-200 μm, and the particles include a mixture of a biologic and at least one dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. 90 The larger the particle size, the higher the percentage of the total amount of biologic released on day 1.

[0275] In such embodiments, the biologic is a recombinant protein, a lipid encapsulating a nucleic acid, or a virus that contains at least one heterologous nucleic acid sequence. In some embodiments, the virus is selected from the group consisting of AAV1, AAV2, such as AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In these embodiments, the AAV contains at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can code for a therapeutic protein, as described in the section under the heading of biologics.

[0276] In another preferred embodiment, the recombinant protein is selected from the group consisting of an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0277] In one embodiment, the biologic is a virus and the total amount of the virus contained in the controlled release pharma- ceutical acceptable implant is at least 10 9 In certain embodiments, the total amount of virus is 10 9 ~10 15 In another preferred embodiment, the total amount of virus is 10 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 vg.

[0278] In one embodiment, the biologic is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In one embodiment, the total amount of AAV contained in the controlled release pharma- ceutically acceptable implant is at least 10 9 In certain embodiments, the total amount of AAV is 10 9 ~10 15 In another preferred embodiment, the total amount of AAV is 10 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 vg.

[0279] In one embodiment, the biologic is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In one embodiment, the total amount of AAV contained in the controlled release pharma- ceutically acceptable implant is at least 10 10 In certain embodiments, the total amount of AAV is 10 10 ~10 15 In another preferred embodiment, the total amount of AAV is 10 10 ~10 15 In another preferred embodiment, the total amount of AAV is 10 10 ~10 14 In another preferred embodiment, the total amount of AAV is 10 10 ~10 13 In another preferred embodiment, the total amount of AAV is 10 10 ~10 12 In another preferred embodiment, the total amount of AAV is 10 10 ~10 11 vg.

[0280] In this embodiment, the controlled release may be characterized as the amount of AAV released on day 1 being 0-50% or less of the total amount of AAV on day 1, such as 0-25%, 0-20%, 0-10%, 0-5%, or about 0%, and the amount of AAV released per day being 50% or less of the total amount of AAV released per day from the second to the last day of the controlled release, and / or the number of days required for 100% release of AAV being 4 days or more. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence may be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it may encode a therapeutic protein, as described in the section under the heading Biologics.

[0281] In this embodiment, the controlled release is such that the amount of AAV released on day 1 is 9.0×10 9 ~1.5×10 10 AAVvg or less released per day from the second to the last day of controlled release of 5.0 × 10 9 ~1.5×10 10 AAVvg, etc., 10 8 , or 10 9 , or 10 10 10 per day, such as orders 11 The AAV may be characterized as being on the order of vgAAV or less and / or requiring 4 days or more for 100% release of AAV. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence may be either a coding or a non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it may encode a therapeutic protein, as described in the section under the heading Biologics.

[0282] In one embodiment, the biologic is a recombinant protein and the total amount of recombinant protein contained in the pharma- ceutically acceptable implant for controlled release is at least 100 μg. In certain embodiments, the total amount of recombinant protein is 100-3000 μg. In other preferred embodiments, the total amount of recombinant protein mixed with at least one dehydration stabilizer is 100-2,500 μg, or 100-2000 μg.

[0283] In another preferred embodiment, the biologic is a recombinant protein such as an antibody, antigen-binding fragment, fusion protein, or hormone.

[0284] Pharmaceutically acceptable implants In accordance with the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel as described in the previous section, a biologic also as described in the previous section, and at least one dehydration stabilizer also as described in the previous section.

[0285] Throughout this section, references to (w / w)% should be construed as being based on the weight of the pharma- ceutically acceptable implant.

[0286] Throughout this section, in all embodiments, including all parameters disclosed in this section, the terms "particle" or "total particles" or "Dv 90 Each of the "particle sizes" is described as relating to particles that include a mixture of a biologic and at least one dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof.

[0287] Throughout this section, in all embodiments, including all parameters disclosed in this section, the term "particle" or "total particles" or "Dv 90 "Particle size" may also refer to the mixture described in the following paragraph.

[0288] In some embodiments, a particle or all particles in an implant of the invention, such as a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic as described above and at least one dehydration stabilizer as described above.

[0289] In some embodiments, an implant of the invention, e.g., a particle or the whole particle in a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0290] In some embodiments, an implant of the invention, e.g., a particle in a pharma- ceutically acceptable implant of the invention, or the entire particle, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, e.g., F-68, F-127, or F123.

[0291] In some embodiments, a particle, or the entire particle, in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0292] In some embodiments, the particles, or the entire particles, in the implants of the invention, e.g., the pharma- ceutically acceptable implants of the invention, are comprised of a mixture of the biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, e.g., F-68, F-127, or F123.

[0293] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, for example, a pharma- ceutically acceptable implant of the invention that includes a mixture of a biologic as described above and at least one dehydration stabilizer as described above.

[0294] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention that includes a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0295] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, comprising a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a mixture of non-ionic surfactants, such as F-68, F-127, or F123.

[0296] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention that comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0297] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, which comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, such as F-68, F-127, or F123.

[0298] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof, the particles being dispersed within the xerogel. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less.

[0299] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The molecular weight between the crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within the xerogel.

[0300] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within the xerogel. The (w / w)% of the total particles comprising the mixture of the biologic and the dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc.

[0301] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma- ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within the xerogel. The (w / w)% of the total number of multi-arm precursors is 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc.

[0302] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof, the particles being dispersed within the xerogel. The ratio of (1) the % (w / w) of the total particles comprising the mixture of the biologic and said dehydration stabilizer to (2) the % (w / w) of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0 or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0, etc. In this context, the % (w / w) is based on the weight of the pharma- ceutically acceptable implant.

[0303] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof, the particles being dispersed within the xerogel. In certain embodiments, Dv 90The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of the biologic and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof.

[0304] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor. In this embodiment, the molar ratio of the first reactive group contained in the second multi-arm precursor to the second reactive group contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40.

[0305] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a primary amine, a second multi-arm precursor comprising succinimidyl succinate, and a third multi-arm precursor comprising succinimidyl glutarate. The pharma-ceutically acceptable implant also comprises a particle comprising a mixture of a biologic and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof. In this embodiment, the molar ratio of the succinimidyl succinate group contained in the second multi-arm precursor to the succinimidyl glutarate group contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40.

[0306] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. The molecular weight between the crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The (w / w)% of the total particles comprising the mixture of biologic and said dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc. The (w / w)% of the total number of multi-arm precursors is 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc. The ratio of (1) the % (w / w) of the total particles comprising the mixture of the biologic and the dehydration stabilizer to (2) the % (w / w) of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0, etc. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor. In this embodiment, the molar ratio of the first reactive group contained in the second multi-arm precursor to the second reactive group contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40.90 The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of the biologic and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof.

[0307] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a primary amine, a second multi-arm precursor comprising succinimidyl succinate, and a third multi-arm precursor comprising succinimidyl glutarate. The pharma- ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. The molecular weight between the crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The (w / w)% of the total particles comprising the mixture of biologic and said dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc. The (w / w)% of the total number of multi-arm precursors is 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc. The ratio of (1) the % (w / w) of the total particles comprising the mixture of the biologic and the dehydration stabilizer to (2) the % (w / w) of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0, etc. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor.In this embodiment, the molar ratio of succinimidyl succinate groups contained in the second multi-arm precursor to succinimidyl glutarate groups contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40. In certain embodiments, Dv. 90 The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of the biologic and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof.

[0308] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. 9 ~10 15 vg order, e.g. 10 9 ~10 13The present invention includes particles comprising a mixture of AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount, such as on the order of vg, and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose or trehalose, a sugar alcohol, such as mannitol, or a combination thereof, the particles being dispersed within a xerogel. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can code for a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0309] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile.9 ~10 15 vg order, e.g. 10 9 ~10 13 In certain embodiments, the particles include a mixture of an AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount, e.g., on the order of vg, and at least one dehydration stabilizer selected from a carbohydrate, e.g., a sugar, e.g., sucrose or trehalose, a sugar alcohol, e.g., mannitol, or a combination thereof, wherein the particles are dispersed within a xerogel. 90 The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of AAV and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding nucleic acid sequence or a non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm. 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0310] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The molecular weight between crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The pharma-ceutically acceptable implant also comprises ... 9 ~10 15 vg order, e.g. 10 9 ~10 13 The present invention also includes particles comprising a mixture of AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount, such as on the order of vg, and at least one dehydration stabilizer selected from carbohydrates, such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, wherein the particles are dispersed within a xerogel. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can code for a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11It is on the order of vg.

[0311] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. 9 ~10 15 vg order, e.g. 10 9 ~10 13 The present invention includes particles comprising a mixture of AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and variants, hybrids, or variants thereof, in a total amount, such as on the order of vg, and at least one dehydration stabilizer selected from carbohydrates, such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within a xerogel. The (w / w)% of the total particle comprising the mixture of AAV and the dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20% to 40%, 20% to 30%, etc. In this context, the (w / w)% is based on the weight of the pharma- ceutically acceptable implant. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or a non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~1013 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0312] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. 9 ~10 15 vg order, e.g. 10 9 ~10 13 The present invention also includes particles comprising a mixture of AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount, such as on the order of vg, and at least one dehydration stabilizer selected from carbohydrates, such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within a xerogel. The (w / w)% of the total number of multi-arm precursors is between 20% and 80%, such as between 35% and 75%, such as between 35% and 65%, 35% and 55%, 35% and 45%, etc. In this context, the (w / w)% is based on the weight of the pharma- ceutically acceptable implant. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it may encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~1015 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0313] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. 9 ~10 15 vg order, e.g. 10 9 ~10 13The particles include a mixture of an AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids or variants thereof, in a total amount, e.g., on the order of vg, and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, e.g., sucrose and trehalose, a sugar alcohol, e.g., mannitol, or a combination thereof, wherein the particles are dispersed within a xerogel. The ratio of (1) the % (w / w) of the total particle comprising the mixture of the AAV and the dehydration stabilizer to (2) the % (w / w) of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0 or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or a non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0314] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises ... second reactive group. 9 ~10 15 vg order, e.g. 10 9 ~10 13 vg order, e.g. 10 9 ~10 13 The particle includes a mixture of AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount, such as on the order of vg, and at least one dehydration stabilizer selected from carbohydrates, such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor. In this embodiment, the molar ratio of the first reactive group contained in the second multi-arm precursor to the second reactive group contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or a non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0315] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a primary amine, a second multi-arm precursor comprising succinimidyl succinate, and a third multi-arm precursor comprising succinimidyl glutarate. The pharma-ceutically acceptable implant also comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a primary amine, a second multi-arm precursor comprising succinimidyl succinate, and a third multi-arm precursor comprising succinimidyl glutarate. 9 ~10 15 vg order, e.g. 10 9 ~10 13The particle includes a mixture of AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount, such as on the order of vg, and at least one dehydration stabilizer selected from carbohydrates, such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. In this embodiment, the molar ratio of succinimidyl succinate groups contained in the second multi-arm precursor to succinimidyl glutarate groups contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or a non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0316] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises ... second reactive group. 9 ~10 15 vg order, e.g. 10 9 ~10 13The particles include a mixture of an AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount, such as on the order of vg, and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. The molecular weight between the crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The % (w / w) of the total particles comprising the mixture of the AAV and the dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc. The % (w / w) of the total number of multi-arm precursors is 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc. The ratio of (1) the % (w / w) of total particles comprising the mixture of the AAV and the dehydration stabilizer to (2) the % (w / w) of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0 or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor. In this embodiment, the molar ratio of the first reactive group contained in the second multi-arm precursor to the second reactive group contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40. 90The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of AAV and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding nucleic acid sequence or a non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm. 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0317] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a primary amine, a second multi-arm precursor comprising succinimidyl succinate, and a third multi-arm precursor comprising succinimidyl glutarate. The pharma-ceutically acceptable implant also comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a primary amine, a second multi-arm precursor comprising succinimidyl succinate, and a third multi-arm precursor comprising succinimidyl glutarate. 9 ~10 15 vg order, e.g. 10 9 ~10 13The particles include a mixture of an AAV selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount, such as on the order of vg, and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. The molecular weight between the crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The % (w / w) of the total particles comprising the mixture of the AAV and the dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc. The % (w / w) of the total number of multi-arm precursors is 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc. The ratio of (1) the % (w / w) of total particles comprising the mixture of the AAV and the dehydration stabilizer to (2) the % (w / w) of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0 or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor. In this embodiment, the molar ratio of succinimidyl succinate groups contained in the second multi-arm precursor to succinimidyl glutarate groups contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40. 90The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of AAV and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding nucleic acid sequence or a non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm. 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0318] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma- ceutically acceptable implant also comprises particles comprising a mixture of recombinant protein and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within the xerogel. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0319] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of recombinant protein and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within the xerogel. In certain embodiments, Dv 90 The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particle comprises a mixture of the recombinant protein and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0320] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The molecular weight between the crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The pharma- ceutically acceptable implant also comprises particles comprising a mixture of recombinant protein and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within the xerogel. In this regard, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone. or hormone.

[0321] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma- ceutically acceptable implant also comprises particles comprising a mixture of recombinant protein and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within the xerogel. The (w / w)% of the total particles comprising the mixture of recombinant protein and said dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20% to 40%, 20% to 30%, etc. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0322] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma- ceutically acceptable implant also comprises particles comprising a mixture of recombinant protein and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof, the particles being dispersed within the xerogel. The (w / w)% of the total number of multi-arm precursors is between 20% and 80%, such as between 35% and 75%, such as between 35% and 65%, 35% and 55%, 35% and 45%, etc. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0323] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile, and optionally a third multi-arm precursor comprising an electrophile. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof, the particles being dispersed within the xerogel. The ratio of (1) the (w / w)% of the total particles comprising the mixture of recombinant protein and said dehydration stabilizer to (2) the (w / w)% of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0 or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0, etc. In this context, the (w / w)% is based on the weight of the pharma- ceutically acceptable implant. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0324] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma- ceutically acceptable implant also comprises a particle comprising a mixture of a recombinant protein and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor. In this embodiment, the molar ratio of the first reactive group contained in the second multi-arm precursor to the second reactive group contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0325] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a primary amine, a second multi-arm precursor comprising succinimidyl succinate, and a third multi-arm precursor comprising succinimidyl glutarate. The pharma-ceutically acceptable implant also comprises a particle comprising a mixture of a recombinant protein and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof. In this embodiment, the molar ratio of the succinimidyl succinate group contained in the second multi-arm precursor to the succinimidyl glutarate group contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0326] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of recombinant protein and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. The molecular weight between the crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The (w / w)% of the total particles comprising the mixture of recombinant protein and said dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc. The (w / w)% of the total number of multi-arm precursors is 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc. The ratio of (1) the (w / w)% of the total particles comprising the mixture of recombinant protein and said dehydration stabilizer to (2) the (w / w)% of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0 or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor. In this embodiment, the molar ratio of the first reactive group contained in the second multi-arm precursor to the second reactive group contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40.In certain embodiments, Dv. 90 The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particle comprises a mixture of the recombinant protein and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0327] According to the present invention, there is provided a pharma- ceutically acceptable implant comprising a xerogel comprising a matrix comprising covalently crosslinked multi-arm precursors comprising a first multi-arm precursor comprising a primary amine, a second multi-arm precursor comprising succinimidyl succinate, and a third multi-arm precursor comprising succinimidyl glutarate. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of recombinant protein and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The total (w / w) % of the dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less. The molecular weight between the crosslinks of the xerogel is 7-25 kDa, 9-20 kDa, or 10-15 kDa. The (w / w)% of the total particles comprising the mixture of recombinant protein and said dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc. The (w / w)% of the total number of multi-arm precursors is 20%-80%, such as 35%-75%, such as 35%-65%, 35%-55%, 35%-45%, etc. The ratio of (1) the (w / w)% of the total particles comprising the mixture of recombinant protein and said dehydration stabilizer to (2) the (w / w)% of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0 or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0. In one embodiment, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor.In this embodiment, the molar ratio of succinimidyl succinate groups contained in the second multi-arm precursor to succinimidyl glutarate groups contained in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40. In certain embodiments, Dv. 90 The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particle comprises a mixture of the recombinant protein and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0328] Other medicaments in the implant that are pharma- ceutically acceptable The pharma- ceutically acceptable implant according to the disclosure of this section may or may not include a "non-functional polymer". The term "non-functional polymer" is defined above and refers to any polymer that does not participate in a cross-linking reaction between precursors. The polymer may be further defined as a polymer that is soluble in both organic solvents and water. In one embodiment, the additional polymer is a non-functional polymer used as a bulking agent in the pharma- ceutically acceptable implant. The MW of the polymer may be 1,000-35,000 Da, such as 5,000-35,000 Da, such as 5,000-10,000 Da, 7,000-10,000 Da, 8,000-15,000 Da, 8,000-25,000 Da, or 5,000 Da or more.

[0329] The polymer may be selected from poly(ethylene) oxide, polyethylene glycol, polyvinylpyrrolidinone, polyvinyl alcohol, polyalkylene oxide, methacrylic acid or other vinyl monomers, acyl chlorides such as methacryloyl chloride, isocyanates, or 2-isocyanatoethyl methacrylate, electrophilic poly(ethylene glycol) methacrylate (PEGMA).

[0330] The additional polymers may be branched (multi-armed) or linear. In the case of branched polymers, the core refers to a continuous portion of the molecule joined to arms extending from the core, the arms often having functional groups at the ends of the branches. It is noted that these polymers may have, for example, 2-100 arms, each arm having a terminus, and some precursors may be dendrimers or other highly branched materials. The arms refer to linear chains of chemical groups connecting the crosslinkable functional groups to the polymer core. In some embodiments, these additional polymers may include 3-300 arms. One of ordinary skill in the art will immediately appreciate that all ranges and values ​​within the explicitly stated ranges are contemplated, for example, 4, 6, 8, 10, 12, 4-16, 8-100, 6, 8, 10, 12, or at least 4 arms.

[0331] In various embodiments, the additional polymer is selected from the group consisting of polyalkylene oxides, such as polyethylene glycol, polyvinylpyrrolidinone, and polyvinyl alcohol.

[0332] Implant morphology Pharmaceutically acceptable implants according to the disclosure in this section may be in the form of a particulate slurry, an in situ gel, a sheet, a film, a rod, or a fiber, each of which represents an embodiment of the invention that may be combined with the disclosure in this section.

[0333] In various embodiments, the pharma- ceutical acceptable implant is in the form of a fiber. The fiber may be characterized by its diameter and / or length. Additionally, the fiber diameter and length may be further characterized as proximal, intermediate, or distal, respectively. The proximal, intermediate, and distal diameters may be the same or different. The proximal, intermediate, and distal lengths may be the same or different. Thus, the term "fiber diameter" refers to the proximal diameter, intermediate diameter, distal diameter, or the average of these three diameters. In some embodiments, the term "fiber diameter" is the average of all three diameters. Thus, the term "fiber length" refers to the proximal length, intermediate length, distal length, or the average of these three lengths. In some embodiments, the term "fiber length" is the average of all three lengths.

[0334] In various embodiments, the fibers are characterized by a diameter of about 0.1 mm or greater and / or a length of about 2.0 mm or greater, hi some embodiments, the fibers are characterized by a diameter of about 0.15 mm or greater and / or a length of about 3.0 mm or greater.

[0335] Total amount or concentration of biologic The total amount or concentration of a biologic contained in a pharma- ceutically acceptable implant varies depending on the type of biologic.

[0336] In one embodiment, the biologic is a virus and is administered in a pharma- ceutically acceptable implant at least 10 9 In certain embodiments, the virus is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 9 ~10 15 In another preferred embodiment, the virus is present in a pharma- ceutical implant in a total amount of 10 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~1012 In some embodiments, the virus is present in a total amount of at least 10 vg. 13 vg / cm 3 , e.g. at least 10 14 vg / cm 3 In these embodiments, the virus comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding nucleic acid sequence or a non-coding nucleic acid sequence. If the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can code for a therapeutic protein, as described in the section under the heading of biologics.

[0337] In one embodiment, the biologic is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, and is administered in a pharmacologic implant in a concentration of at least 10 9 In certain embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 9 ~10 15 In another preferred embodiment, the AAV is present in a pharma- ceutical implant in a total amount of 10 vg. 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 In some embodiments, the AAV comprises at least 10 13 vg / cm 3 , e.g. at least 10 14 vg / cm 3In some embodiments, the AAV is included in the pharma- ceutically acceptable implant at a total concentration of 10. In these embodiments, the AAV includes at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10. 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0338] In one embodiment, the biologic is a recombinant protein and is present in the pharma- ceutically acceptable implant in a total amount of at least 10 μg. In certain embodiments, the recombinant protein is present in the pharma-ceutically acceptable implant in a total amount of 10-3000 μg. In other preferred embodiments, the recombinant protein is present in the pharma-ceutically acceptable implant in a total amount of 10-2,500 μg, or 10-2000 μg.

[0339] In one embodiment, the biologic is an antibody and is present in the pharmaceutically acceptable implant in a total amount of at least 100 μg. In a particular embodiment, the antibody is present in the pharmaceutically acceptable implant in a total amount of 100-3000 μg. In another preferred embodiment, the antibody is present in the pharmaceutically acceptable implant in a total amount of 300-3000 μg.

[0340] In one embodiment, the biologic is an anti-VEGF antibody, such as ranibizumab, and is present in the pharmaceutically acceptable implant in a total amount of at least 500 μg. In a particular embodiment, the ranibizumab is present in the pharmaceutically acceptable implant in a total amount of 500-1000 μg. In another preferred embodiment, the ranibizumab is present in the pharmaceutically acceptable implant in a total amount of 300-1000 μg.

[0341] In one embodiment, the biologic is an anti-VEGF antibody, such as bevacizumab, and is present in the pharmaceutically acceptable implant in a total amount of at least 1,500 μg. In certain embodiments, the bevacizumab is present in the pharmaceutically acceptable implant in a total amount of 1,500-3,000 μg. In other preferred embodiments, the bevacizumab is present in the pharmaceutically acceptable implant in a total amount of 1,500-2,000 μg, or 1,250 μg.

[0342] In one embodiment, the biologic is a fusion protein such as aflibercept, and is included in the pharmaceutically acceptable implant in a total amount of at least 2000 μg. In a particular embodiment, the aflibercept is included in the pharmaceutically acceptable implant in a total amount of 2,000-3,000 μg. In another preferred embodiment, the aflibercept is included in the pharmaceutically acceptable implant in a total amount of 2,000 μg.

[0343] In one embodiment, a pharma- ceutically acceptable implant of the invention is provided, characterized in that the implant induces an immune response, such as an adaptive immune response, such as a humoral immune response, to the biologic contained in the implant, as measured by a detectable serum ADA titer in rabbits. The detectable serum ADA titer is 20,000 or less, or 15,000 or less, or 10,000 or less, or 8,000 or less, or 7,000 or less, or 5,000 or less, or 2,000 or less, or 1,000 or less, or below the limit of detection, compared to the serum titer of ADA at baseline in rabbits. In one embodiment, the implant is administered to the eye of the rabbit, such as by intravitreal administration. In one embodiment, the serum titer of ADA relates to any time point between 8 weeks and 13 weeks after administration, such as 8 weeks after administration or 13 weeks after administration. In one embodiment, the serum titer of ADA relates to the corresponding time point compared to 8 weeks or 13 weeks when the biologic is AAV2.7m8. In one embodiment, the biologic is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In one embodiment, the AAV is at least 10% in the pharma- ceutically acceptable implant. 9 In certain embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 9 ~10 15 In another embodiment, the AAV is present in a pharma- ceutical acceptable implant in a total amount of 10 vg. 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 In some embodiments, the AAV comprises at least 10 13 vg / cm 3 , e.g. at least 10 14 vg / cm 3In one embodiment, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or a non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0344] In one embodiment, a pharma- ceutically acceptable implant of the invention is provided, characterized in that the implant provides a detectable total amount of the biologic that is at least 4 logs less, 5 logs less, 6 logs less, 7 logs less, 8 logs less, 9 logs less, or below the detection limit per mL of rabbit plasma, relative to the total amount of the biologic contained in the implant. In one embodiment, the implant is administered to the rabbit eye, such as by intravitreal administration. In one embodiment, the detectable total concentration of the biologic relates to any time point from day 1 to day 3 after administration, such as day 2 after administration. In one embodiment, the detectable total concentration of the biologic relates to a corresponding time point compared to any time point from day 1 to day 3 after administration, such as day 2 after administration, when the biologic is AAV2.7m8. In one embodiment, the biologic is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In one embodiment, the AAV is at least 10% in the pharma- ceutically acceptable implant. 9 In certain embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 9 ~10 15 In another embodiment, the AAV is present in a pharma- ceutical acceptable implant in a total amount of 10 vg. 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 In some embodiments, the AAV comprises at least 10 13 vg / cm 3 , e.g. at least 10 14 vg / cm 3In one embodiment, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or a non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11In one embodiment, a pharma- ceutically acceptable implant is provided comprising an AAV comprising a heterologous nucleic acid sequence, characterized in that the implant provides a detectable total amount of heterologous nucleic acid sequence that is at least 4 log less, 5 log less, 6 log less, 7 log less, 8 log less, 9 log less, or below the detection limit per mL of rabbit plasma, relative to the total amount of AAV contained in the implant. In one embodiment, the implant is administered to the eye of a rabbit, such as by intravitreal administration. In one embodiment, the detectable total concentration of the heterologous nucleic acid sequence relates to any time point from day 1 to day 3 after administration, such as day 2 after administration. In one embodiment, the detectable total concentration of the heterologous nucleic acid sequence relates to a corresponding time point compared to any time point from day 1 to day 3 after administration, such as day 2 after administration, when the biologic is AAV2.7m8. In one embodiment, the biologic is an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof. In one embodiment, the AAV is at least 10% in the pharma- ceutically acceptable implant. 9 In certain embodiments, the AAV is present in a pharma- ceutically acceptable implant in a total amount of 10 vg. 9 ~10 15 In another embodiment, the AAV is present in a pharma- ceutical acceptable implant in a total amount of 10 vg. 9 ~10 15 vg, 10 9 ~10 13 vg, or 10 9 ~10 12 In some embodiments, the AAV comprises at least 10 13 vg / cm 3 , e.g. at least 10 14 vg / cm 3In one embodiment, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or a non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0345] In one embodiment, an implant, such as a pharma- ceutically acceptable implant, of the present invention does not include a biologic adsorbed to silica particles, such as mesoporous silica particles, or equivalents.

[0346] In one embodiment, an implant, such as a pharma- ceutically acceptable implant, of the present invention does not include a biologic that adsorbs to a fatty acid moiety or its equivalent.

[0347] In one embodiment, an implant, such as a pharma- ceutically acceptable implant, of the invention does not comprise AAV adsorbed to silica particles, such as mesoporous silica particles or equivalents.

[0348] In one embodiment, an implant, such as a pharma- ceutically acceptable implant, of the invention does not comprise an AAV adsorbed to a fatty acid moiety or equivalent thereof.

[0349] Pharmaceutically acceptable implants for the controlled release of biologics - Patent Application 20070229633 In one aspect of the present invention, a pharma- ceutically acceptable implant is provided for controlled release of the total amount of a biologic contained therein. Throughout this section, controlled release shall be considered as controlled release measured under physiological conditions, such as pH 7.2-7.4 and temperature 37° C., from the time the implant is first immersed in an aqueous solution under those conditions. After exposure to physiological conditions, the xerogel contained in the pharma-ceutically acceptable implant forms a hydrogel.

[0350] Throughout this section, references to (w / w)% should be construed as being based on the weight of the pharma- ceutically acceptable implant.

[0351] Throughout this section, in all embodiments, including all parameters disclosed in this section, the terms "particle" or "total particles" or "Dv 90 Each of the "particle sizes" is described as relating to particles that include a mixture of a biologic and at least one dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof.

[0352] Throughout this section, in all embodiments, including all parameters disclosed in this section, the term "particle" or "total particles" or "Dv 90 "Particle size" may also refer to the mixture described in the following paragraph.

[0353] In some embodiments, a particle or all particles in an implant of the invention, such as a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic as described above and at least one dehydration stabilizer as described above.

[0354] In some embodiments, an implant of the invention, e.g., a particle or the whole particle in a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0355] In some embodiments, an implant of the invention, e.g., a particle in a pharma- ceutically acceptable implant of the invention, or the entire particle, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, e.g., F-68, F-127, or F123.

[0356] In some embodiments, a particle, or the entire particle, in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0357] In some embodiments, the particles, or the entire particles, in the implants of the invention, e.g., the pharma- ceutically acceptable implants of the invention, are comprised of a mixture of the biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, e.g., F-68, F-127, or F123.

[0358] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, for example, a pharma- ceutically acceptable implant of the invention that includes a mixture of a biologic as described above and at least one dehydration stabilizer as described above.

[0359] In some embodiments, Dv 90Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention that includes a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0360] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, comprising a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a mixture of non-ionic surfactants, such as F-68, F-127, or F123.

[0361] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention that comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer or surfactant.

[0362] In some embodiments, Dv 90 Particle size, such as particle size, refers to particles in an implant of the invention, e.g., a pharma- ceutically acceptable implant of the invention, which comprises a mixture of a biologic, at least one dehydration stabilizer, and at least one other stabilizer, such as a buffer, such as PBS, containing at least two or at least three salts, and a non-ionic surfactant, such as F-68, F-127, or F123.

[0363] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 2 days.

[0364] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 3 days.

[0365] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 4-7 days.

[0366] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 10-15 days.

[0367] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 10-30 days.

[0368] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein, which may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is greater than 30 days.

[0369] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is 5 weeks or more.

[0370] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein, which may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is about 6 weeks.

[0371] According to the present invention, a controlled release is characterized in that the amount of biologic released on day 1 is 0-25%, 0-20%, 0-10%, 0-5%, or about 0% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release is 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 3 days, but not more than 30 days, not more than 25 days, or not more than 16 days.

[0372] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 2 days. The controlled release may be characterized as the amount of biologic released on day 1 being 0-25% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 2 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-20% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 2 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 2 days. In this context, a biologic may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 2 days. 9 From 10 15 In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 1010 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0373] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 3 days. The controlled release may be characterized as the amount of biologic released on day 1 being 0-25% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 3 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-20% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 3 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 3 days. In this context, a biologic may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 3 days.9 From 10 15 In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0374] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 5-7 days. The controlled release may be characterized as the amount of biologic released on day 1 being 0-25% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 5-7 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-20% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 5-7 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 5-7 days. In this context, a biologic may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 5-7 days. 9 From 10 15In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0375] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 10-15 days. The controlled release may be characterized as the amount of biologic released on day 1 being 0-25% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 10-15 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-20% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 10-15 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 10-15 days. In this context, a biologic may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 10-15 days. 9 From 10 15In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0376] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 10-30 days. The controlled release may be characterized as the amount of biologic released on day 1 being 0-25% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 10-30 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-20% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 10-30 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 10-30 days. In this context, a biologic may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is at least 10-30 days. 9 From 10 15In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0377] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required to release 100% of the total amount of the biologic is greater than 30 days. The controlled release may be characterized as the amount of biologic released on day 1 being 0-25% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required to release 100% of the total amount of the biologic is greater than 30 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-20% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is greater than 30 days. A controlled release may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is greater than 30 days. In this context, a biologic may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is greater than 30 days. 9 From 10 15 In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0378] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is 5 weeks or more. The controlled release may be characterized as the amount of biologic released on day 1 being 0-25% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is 5 weeks or more. A controlled release may be characterized by the amount of biologic released on day 1 being 0-20% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is 5 weeks or more. A controlled release may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is 5 weeks or more. In this context, a biologic may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is 5 weeks or more. 9 From 10 15In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0379] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of a total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on day 1 being 0-50% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is about 6 weeks. The controlled release may be characterized as the amount of biologic released on day 1 being 0-25% of the total amount of the biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is about 6 weeks. A controlled release may be characterized by the amount of biologic released on day 1 being 0-20% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is about 6 weeks. A controlled release may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is about 6 weeks. In this context, a biologic may be characterized by the amount of biologic released on day 1 being 0-10% of the total amount of biologic, the amount of biologic released per day from day 2 through the last day of release being 0-50% of the total amount of biologic, and / or the number of days required to release 100% of the total amount of biologic is about 6 weeks. 9 From 10 15 In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0380] According to the present invention, a controlled release is characterized in that the amount of biologic released on the first day is 0-25%, 0-20%, 0-10%, 0-5%, or about 0% of the total amount of biologic, the amount of biologic released per day from the second to the last day of release is 0-50% of the total amount of biologic, and / or the number of days required for 100% release of the total amount of biologic is at least 3 days, but not more than 6 weeks, not more than 5 weeks, not more than 30 days, not more than 25 days, or not more than 16 days. 9 From 10 15 In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, e.g., AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof, in a total amount on the order of 10 vg. In these embodiments, the AAV comprises at least one heterologous nucleic acid sequence. In these embodiments, the heterologous nucleic acid sequence can be either a coding or non-coding nucleic acid sequence. When the heterologous nucleic acid sequence is a coding nucleic acid sequence, it can encode a therapeutic protein, as described in the section under the heading Biologics. In some embodiments, the total amount of AAV is on the order of 10 10 ~10 15 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 14In some embodiments, the total amount of AAV is on the order of 10 10 ~10 13 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 12 In some embodiments, the total amount of AAV is on the order of 10 10 ~10 11 It is on the order of vg.

[0381] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on the first day being 0-50% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 2 days. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0382] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on the first day being 0-50% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 3 days. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0383] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on the first day being 0-50% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 5-7 days. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0384] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on the first day being 0-50% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 10-15 days. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0385] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on the first day being 0-50% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 10-30 days. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0386] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on the first day being 0-50% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is greater than 30 days. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0387] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on the first day being 0-50% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is 5 weeks or more. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0388] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release may be characterized as the amount of biologic released on the first day being 0-50% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release being 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is about 6 weeks. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0389] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The controlled release is characterized in that the amount of biologic released on day 1 is 0-25%, 0-20%, 0-10%, 0-5%, or about 0% of the total amount of the biologic, the amount of biologic released per day from the second to the last day of release is 0-50% of the total amount of the biologic, and / or the number of days required for 100% release of the total amount of the biologic is at least 3 days, but not more than 6 weeks, not more than 5 weeks, not more than 30 days, not more than 25 days, or not more than 16 days. In this context, the recombinant protein may be an antibody, an antigen-binding fragment, a fusion protein, a cytokine, a transcription factor, an enzyme, or a hormone.

[0390] In one embodiment, the controlled release having the above characteristics includes a zero order release, e.g., near zero order release, or substantially zero order release, etc. In one embodiment, the zero order release, near zero order release, or substantially zero order release begins at least 1 day after the pharma- ceutically acceptable implant is immersed under physiological conditions, such as pH 7.2-7.4 and 37°C.

[0391] A dosage form exhibiting a zero-order release rate will exhibit a relatively straight line in a graphical representation of the percentage of biologic released versus time. In certain embodiments of the present invention, zero-order release is achieved throughout the entire release period. In certain embodiments of the present invention, zero-order release is achieved throughout a portion of the release period. In certain such embodiments, zero-order release is achieved from the end of the first day, i.e., from 24 hours after release begins, until release ends. If there is little or no release by the end of the first day, such release is considered to have had a lag time of one day or 24 hours. Such lag times may also be longer. If there is a large amount of release by the end of the first day, such release is considered to have had a burst within the first day or within 24 hours. Such bursts may also be longer. Zero-order release may also be achieved during the entire release period. In this context, the entire release period is defined as until 95% of release is achieved.

[0392] Within the meaning of the present invention, zero-order release is defined as being achieved when the release during each time period is proportional to the elapsed time. Proportional to the elapsed time means that the proportional release is calculated based on the total time of zero-order release that defines a line (the release of the % cumulative release during the entire period that zero-order is achieved divided by the total time that defines the line), and the release at any point in between, i.e., between the start of zero-order release and the end of zero-order release, is within 20% points of the % cumulative release of the proportional release defined by the line.

[0393] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The pharma-ceutically acceptable implant comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and optionally a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof. The inventors have discovered that the total (w / w) % of carbohydrate, sugar alcohol, or a combination thereof in the pharma-ceutically acceptable implant allows for controlled release. In this regard, the total (w / w) % of said dehydration stabilizer is 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less, or 30% or less.

[0394] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The pharma-ceutically acceptable implant comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and optionally a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The inventors have found that Dv 90 Particle Size or Dn 90 D such as particle size 90 It has been discovered that the particle size allows for controlled release. 90The particle size is between 10 μm and 200 μm, such as between 35 μm and 75 μm, such as between 35 μm and 100 μm, or between 35 μm and 150 μm, and the particles comprise a mixture of the biologic and said dehydration stabilizer, such as a carbohydrate, a sugar alcohol, or a combination thereof.

[0395] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The pharma-ceutically acceptable implant comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and optionally a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The inventors have discovered that the molecular weight between the crosslinks of the xerogel contained in the pharma-ceutically acceptable implant allows for controlled release. The controlled release provided by this feature is as described above in this section and relates to the release of the biologic on day 1, the release per day thereafter, and the total number of days required for 100% release. In this regard, the molecular weight between crosslinks of the xerogel is between 7 and 25 kDa, between 9 and 20 kDa, or between 10 and 15 kDa.

[0396] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlling the release of the total amount of a biologic contained therein. The pharma-ceutically acceptable implant comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and optionally a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof. The inventors have discovered that the (w / w)% of the total particles comprising a mixture of a biologic and a carbohydrate, a sugar alcohol, or a combination thereof in the pharma-ceutically acceptable implant allows for controlled release. The controlled release provided by this feature is as described above in this section and relates to the release of the biologic on day 1, the release per day thereafter, and the total number of days required for 100% release. In this regard, the % (w / w) of the total particles comprising the mixture of the biologic and said dehydration stabilizer is 80% or less, or 70% or less, or 60% or less, or 50% or less, such as 20%-40%, 20%-30%, etc.

[0397] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlling the release of the total amount of a biologic contained therein. The pharma-ceutically acceptable implant comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and optionally a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The inventors have discovered that a (w / w)% of the total number of multi-arm precursors in the pharma-ceutically acceptable implant allows for controlled release. The controlled release provided by this feature is as described above in this section and relates to the release of the biologic on day 1, the release per day thereafter, and the total number of days required for 100% release. In this regard, the (w / w)% of the total number of multi-arm precursors may be between 20% and 80%, such as between 35% and 75%, for example between 35% and 65%, 35% and 55%, 35% and 45%, etc.

[0398] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The pharma-ceutically acceptable implant comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and optionally a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from a carbohydrate, such as a sugar, such as sucrose and trehalose, a sugar alcohol, such as mannitol, or a combination thereof. The inventors have discovered that the ratio of the (w / w)% of the total particles comprising a mixture of a biologic and a carbohydrate, a sugar alcohol, or a combination thereof in the pharma-ceutically acceptable implant to the (w / w)% of the total number of multi-arm precursors in the pharma-ceutically acceptable implant allows for controlled release. The controlled release provided by this feature is as described previously in this section and relates to the release of the biologic on day 1, the daily release thereafter, and the total number of days required for 100% release. In this regard, the ratio of (1) the % (w / w) of the total particles comprising the mixture of the biological agent and said dehydration stabilizer to (2) the % (w / w) of the total number of multi-arm precursors is 0.3-4.0, such as 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0 or 0.5-4.0, such as 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, or 0.6-4.0, 0.6-3.5, 0.6-3.0, 0.6-2.5, 0.6-2.0, 0.6-1.5, or 0.6-1.0.

[0399] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlling the release of the total amount of a biologic contained therein. The pharma- ceutically acceptable implant comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma- ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The molecular weight between the crosslinks of the xerogel contained in the pharma- ceutical acceptable implant allows for the controlled release. The inventors have discovered that the molar ratio of the first reactive group contained in the second multi-arm precursor in the pharma- ceutical acceptable implant to the second reactive group contained in the third multi-arm precursor in the pharma- ceutical acceptable implant allows for the controlled release. The controlled release provided by this feature is as described above in this section and relates to the release of the biologic on day 1, the release per day thereafter, and the total number of days required for 100% release. In this regard, the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor. In this regard, the molar ratio of the first reactive group in the second multi-arm precursor to the second reactive group in the third multi-arm precursor is 30-90:70-10, 40-80:60-20, 50-70:50-30, or 40-60:60-40.

[0400] According to the present invention, there is provided a pharma- ceutically acceptable implant for controlled release of the total amount of a biologic contained therein. The pharma-ceutically acceptable implant comprises a xerogel comprising a matrix comprising a covalently crosslinked multi-arm precursor comprising a first multi-arm precursor comprising a nucleophile, a second multi-arm precursor comprising an electrophile comprising a first reactive group, and optionally a third multi-arm precursor comprising an electrophile comprising a second reactive group. The pharma-ceutically acceptable implant also comprises particles comprising a mixture of a biologic and at least one dehydration stabilizer selected from carbohydrates such as sugars, such as sucrose and trehalose, sugar alcohols, such as mannitol, or combinations thereof. The inventors have discovered that when the desired controlled release is characterized by the number of day...

Claims

1. 1. A pharmaceutically acceptable implant comprising a xerogel, a biologic, and at least one dehydration stabilizer, The pharmaceutically acceptable implant, wherein the biologic is a virus or virus-like particle, and the virus or virus-like particle comprises a viral nucleic acid and one or more heterologous nucleic acids.

2. 10. The pharmaceutically acceptable implant of claim 1, wherein the xerogel comprises a matrix comprising covalently crosslinked multi-arm precursors, having dispersed therein particles comprising a mixture of a biologic, at least one dehydration stabilizer, and optionally at least one additional stabilizer.

3. 10. The pharmaceutically acceptable implant of claim 1, wherein the at least one dehydration stabilizer is a carbohydrate, a sugar alcohol, or a combination thereof.

4. 3. The pharmaceutically acceptable implant of claim 2, wherein the multi-arm precursor comprises at least two multi-arm precursors, including a first multi-arm precursor comprising a first functional group and a second multi-arm precursor comprising a second functional group.

5. 5. The pharmaceutically acceptable implant of claim 4, wherein the multi-arm precursor comprises a third multi-arm precursor that includes the same functional group as the second multi-arm precursor.

6. 5. The pharmaceutically acceptable implant of claim 4, wherein each of the first functional group and the second functional group is selected from the group consisting of an electrophile and a nucleophile, and the reaction between the first functional group and the second functional group is an electrophile-nucleophile reaction that forms a covalent bond.

7. 7. The pharmaceutically acceptable implant of claim 6, wherein the nucleophile comprises an amine, a thiol, or a hydrazide.

8. 7. The pharmaceutically acceptable implant of claim 6, wherein the electrophile comprises a succinimidyl ester, succinimidyl carbonate, nitrophenyl carbonate, aldehyde, ketone, acrylate, acrylamide, maleimide, vinyl sulfone, iodoacetamide, alkene, alkyne, norbornene, epoxide, mesylate, tosylate, tresyl, cyanurate, orthopyridyl disulfide, or halide.

9. 4. The pharmaceutically acceptable implant of claim 3, wherein the carbohydrate is selected from a monosaccharide, a disaccharide, an oligosaccharide, a water-soluble polysaccharide, or any combination thereof.

10. 10. The pharmaceutically acceptable implant of claim 9, wherein the carbohydrate is a sugar.

11. 11. The pharmaceutically acceptable implant of claim 10, wherein the sugar is sucrose, trehalose, raffinose, stachyose, verbascose, hydrates thereof, and any combination thereof.

12. 4. The pharmaceutically acceptable implant of claim 3, wherein the sugar alcohol is selected from the group consisting of erythritol, glycerol, isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol, and combinations thereof.

13. 2. The pharmaceutically acceptable implant of claim 1, wherein the implant is in the form of a fiber, the fiber having a diameter of 0.1 mm or more and / or a length of 2.0 mm or more.

14. The implant for controlled release of a total amount of the biologic, wherein the controlled release comprises: (A) the amount of the biologic released on day 1 is 0-50% of the total amount of the biologic; (B) the amount of the biologic released per day from the second to the last day of release is 0-50% of the total amount of the biologic; and / or (C) the number of days required for 100% release of the total amount of the biologic is at least 2 days; 10. The pharmaceutically acceptable implant of claim 1, wherein the controlled release is measured under physiological conditions from the time the implant is first immersed in an aqueous solution under those conditions.

15. The pharmaceutically acceptable implant of claim 3, wherein the implant is for controlled release of the total amount of the biological agent, the controlled release being characterized by the number of days required for 100% release of the total amount of the biological agent, which number of days is at least 2 days, and the total (w / w) percentage of the carbohydrate, sugar alcohol, or combination thereof is 60% or less based on the weight of the pharmaceutically acceptable implant.

16. The multi-arm precursor of claim 1, (i) a first multi-arm precursor comprising the nucleophile of claim 7; (ii) a second multi-arm precursor comprising the electrophile of claim 8 that comprises a first reactive group; and 10. The pharmaceutically acceptable implant of claim 2, comprising at least three multi-arm precursors, including (iii) a third multi-arm precursor comprising the electrophile of claim 8, wherein the electrophile comprises a second reactive group.

17. 17. The pharmaceutically acceptable implant of claim 16, wherein the third multi-arm precursor has a longer hydrolysis half-life compared to the second multi-arm precursor.

18. 17. The pharmaceutically acceptable implant of claim 16, wherein the electrophile-nucleophile reaction between the first multi-arm precursor and the second multi-arm precursor, and between the first multi-arm precursor and the third multi-arm precursor, forms the covalent bond.

19. (i) the first multi-arm precursor comprises a primary amine; (ii) the second multi-arm precursor comprises a succinimidyl ester that includes a first reactive group; (iii) the third multi-arm precursor comprises a succinimidyl ester that includes a second reactive group; 17. The pharmaceutically acceptable implant of claim 16, wherein the first reactive group is succinimidyl succinate and the second reactive group is succinimidyl glutarate.

20. 2. The pharmaceutically acceptable implant of claim 1, wherein each heterologous nucleic acid is selected from the group consisting of DNA and RNA.

21. 21. The pharmaceutically acceptable implant of claim 20, wherein the heterologous nucleic acid is a non-coding nucleic acid selected from the group consisting of ssDNA (single-stranded DNA), dsDNA (double-stranded DNA), small interfering RNA (siRNA), microRNA, dsRNA, lncRNA, piRNA, rmRNA, sRNA, tiRNA, eRNA, snoRNA, snRNA, circRNA (circular RNA), RNA aptamer, antisense oligonucleotide, guide RNA, tRNA, or any combination thereof.

22. 21. The pharmaceutically acceptable implant of claim 20, wherein the heterologous nucleic acid comprises a coding nucleic acid sequence.

23. 23. The pharmaceutically acceptable implant of claim 22, wherein the coding nucleic acid sequence encodes a therapeutic protein.

24. 2. The pharmaceutically acceptable implant of claim 1, wherein the virus is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus (AAV), lentivirus, and herpes simplex virus.

25. 25. The pharmaceutically acceptable implant of claim 24, wherein the adeno-associated virus (AAV) is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and mutants, hybrids, or variants thereof.

26. The total amount of AAV contained in the implant is at least 10 ９ 25. The pharmaceutically acceptable implant of claim 24, wherein the dosage is on the order of vg.

27. A method for producing a pharmaceutically acceptable implant according to any one of claims 1 to 15 and 17 to 26, comprising: (A) forming an organogel comprising a virus or virus-like particle, the forming comprising forming a matrix comprising at least two covalently crosslinked multi-arm precursors in an organic solvent in the presence of the virus or virus-like particle; (B) forming a xerogel, the forming including removing the organic solvent.

28. A pharmaceutically acceptable implant as described in any of claims 1 to 15 and 17 to 26 for use in a method for treating an eye disease, comprising administering the pharmaceutically acceptable implant to a subject.

29. The pharmaceutically acceptable implant of claim 28, wherein the treatment comprises intravitreal injection of the pharmaceutically acceptable implant into the subject.

30. 29. The pharmaceutically acceptable implant of claim 28, wherein the pharmaceutically acceptable implant is for controlled release of a total amount of adeno-associated virus (AAV), and when the total dose of AAV contained in the pharmaceutically acceptable implant is on the order of less than 2.0 x 10<10> vg, the number of days required for 100% release of the AAV is at least 4 days.

31. The pharmaceutically acceptable implant of claim 28, wherein the pharmaceutically acceptable implant is for controlled release of a total amount of adeno-associated virus (AAV), and when the total dose of AAV contained in the pharmaceutically acceptable implant is on the order of more than 2.0 x 10 10 vg, the number of days required for 100% release of the AAV is at least 7 days.

32. The ocular disease is retinal neovascularization, choroidal neovascularization, exudative AMD, dry AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, corneal transplant rejection, retinoblastoma, melanoma, glaucoma, autoimmune uveitis, uveitis, proliferative vitreoretinopathy, and corneal degeneration, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, macular edema, acute multifocal platelet pigment epitheliopathy, Behcet's disease, birdshot retinochoroidopathy, and posterior uveitis. , posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusion, central retinal vein occlusion, disseminated intravascular coagulation, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal artery microaneurysms, Coats disease, parafoveal telangiectasia, hemiretinal vein occlusion, papillary vasculitis, carotid artery disease (CAD), glaucoma vasculitis, sickle cell retinopathy, angioid streaks, familial exudative vitreoretinopathy, I Hayes' disease, proliferative vitreoretinopathy, diabetic retinopathy, retinal diseases associated with tumors, congenital hypertrophy of the retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, combined hamartoma of the retina and retinal pigment epithelium, retinoblastoma, angioproliferative tumor of the fundus, retinal astrocytoma, intraocular lymphoma, myopic retinal degeneration, acute retinal pigment epitheliitis, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal cancer, retinitis pigmentosa, Leber's disease 29. The pharmaceutically acceptable implant of claim 28, wherein the condition is selected from congenital amaurosis, congenital choroideremia, X-linked retinitis pigmentosa, Best vitelliform macular dystrophy, X-linked retinoschisis, CNGA3 color vision deficiency, CNGB3 color vision deficiency, LHON, Stargardt disease, Usher syndrome, Norrie disease, Bardet-Biedl syndrome, Goldmann-Fabre syndrome, rod-cone dystrophy, Best disease, and red-green color blindness.

33. The pharmaceutically acceptable implant of claim 28, wherein the ocular disease is an ocular genetic disease selected from the group consisting of retinitis pigmentosa, Leber's congenital amaurosis, congenital choroideremia, X-linked retinitis pigmentosa, Best vitelliform macular dystrophy, X-linked retinoschisis, CNGA3 color vision deficiency, CNGB3 color vision deficiency, LHON, Stargardt disease, Usher syndrome, Norrie disease, Bardet-Biedl syndrome, and red-green color blindness.

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