BACKGROUND OF THE INVENTION Field of the Invention

[001] The present disclosure relates to cell culture mediums and formulation feeds for cell culture mediums that are used for recombinant gene therapy (GT) virus production, such as recombinant adeno-associated virus (rAAV), as well as a method of generating recombinant GT virus such as rAAV, and host cells having vectors for recombinant GT virus or rAAV production in culture with the cell culture mediums.

Description of the Background Art

[002] AAV is a small, replication-defective, non-enveloped animal virus that infects humans and some other primate species. Several features of AAV make this virus an attractive vehicle for delivery of therapeutic proteins by gene therapy, including, for example, that AAV is not known to cause human disease and induces a mild immune response, and that AAV can infect both dividing and quiescent cells without integrating into the host cell genome.

[003] AAV has a capsid that includes VP1, VP2, and VP3 proteins, which, in the native viral genome, are produced at the appropriate ratio by alternate splicing of the cap gene and alternate translation initiation at non-AUG start codons. The AAV Rep gene encodes proteins (Rep68, Rep78, Rep40, and Rep52), which are thought essential for regulating viral replication of the native virus in known host cells. An alternative open reading frame (ORF) in the Cap gene encodes the assembly activating protein (AAP), thought to promote capsid assembly, possibly by targeting VP proteins to the host cell nucleolus for capsid assembly. (Sonntag et ah, Proc Natl Acad Sci USA 2010, 107(22): 10220-10225). According to Sonntag, AAP stimulates transport of unassembled VP proteins to the host cell nucleolus for capsid assembly and the nucleolus may provide factors used in the assembly process and proposes chaperones, nucleophosmin, and nucleolin as candidates for such factors. [004] The AAV viral genome is a single stranded deoxyribonucleic acid (DNA) of about 4.7 kb with two 145 nucleotide (nt) inverted terminal repeats (ITRs). The virus relies on cellular proteins for genome replication, including polymerase.

[005] AAV has been successfully produced in mammalian cells lines, such as HEK293 cells, and in insect cells using baculovirus. See Smith et al. Molecular Therapy (2009) 17 11, 1888— 1896 for a discussion of insect cell/baculovirus systems. For use in gene therapy, the Rep and Cap genes can be expressed in trans, thus increasing the size of heterologous genes that can be packaged into the AAV vector. A helper virus or helper virus gene products produced recombinantly by the host cell is generally used for AAV production. Adenovirus and Herpes simplex virus are typically used. Alternatively, helper factors are produced by host cell transgenes. Adenovirus helper factors include El A, E1B, E2A, E40RF6, and VA. In the case of HEK293 cells, the cells already have the ElA/Elb gene, so helper factors E2A, E40RF6 and VA ribonucleic acids (RNAs) are provided as transgenes. See, for example, U.S. Patent Application Publication No. 2014/0377224. AAV are known to exist in a variety of serotypes (e.g., AAV1- AAV13).

[006] Other viruses used for gene therapy include retrovirus, lentivirus, adenovirus, and herpes simplex virus.

[007] For example, recombinant lentiviruses (rLVs) are useful for delivering heterologous transgenes (i.e., genes that are not native to the lentivirus (LV)) to hematopoietic stem cells in order to treat genetic diseases such as adenosine deaminase deficiency (Farinelli, et al, 2014), b- thalassemia, sickle cell disease (Negre et al., 2016), severe combined immune deficiencies, metachromatic leukodystrophy, adrenoleukodystrophy, Wiskott-Aldrich syndrome, chronic granulomatous disease (Booth et al., 2016), and several lysosomal storage disorders (Rastall, et al., 2015).

[008] The LV genus belongs to the Retroviridae family. Characterized by a long incubation period before disease onset in a host, LV received its name from the latin “ lente ”, meaning “slow” (Milone and O’Doherty, Leukemia 32,1529-1541, 2018). Through extensive optimization of lentiviral capsids, the successful production of nonpathogenic lentiviral vectors, which are not capable of replication after initial gene delivery, has established the safety and efficiency of LV vector biotherapy. Most of these vectors are based on human immunodeficiency virus (HIV), which consists of two single-stranded RNA copies coated by 2,000 p24 proteins arranged into a conical capsid (Milone and O’Doherty, Leukemia 32,1529-1541, 2018). Capsid integrity is further protected by a pl7 matrix, all of which is enveloped by a roughly spherical -120 nm diameter phospholipid envelope (Milone and O’Doherty, Leukemia 32,1529-1541, 2018). The large Megadalton size-range of these viral particles enables them to encapsulate a large genome up to 8.5kb (Milone and O’Doherty, Leukemia 32,1529-1541, 2018). This single-stranded RNA genome codes for nine genes, five indispensable for viral survival and function and four accessory genes, flanked by long terminal repeats (LTRs) (Milone and O’Doherty, Leukemia 32,1529-1541, 2018). In recombinant LV vectors designed for gene therapy purposes, the phospholipid envelope is replaced most often by a vesicular stomatitis virus G (VSV-G) envelope, which recognizes the ubiquitously-expressed lipoprotein (LDL) receptor, thus enabling vector transduction in a diverse range of cell types (Milone and O’Doherty, Leukemia 32,1529- 1541, 2018). Additionally, viral accessory genes are removed to enhance vector safety and are replaced by a therapeutic gene expression cassette. The general infection process involves cellular entry by receptor-mediated endocytosis or membrane fusion following attachment of glycoproteins on the vector envelope with their respective cell-membrane receptors (Milone and O’Doherty, Leukemia 32,1529-1541, 2018). Cellular entry is followed by genome-uncoating and reverse transcription of the released single stranded ribonucleic acid (ssRNA). The newly synthesized cDNA is then transported to the nucleus where it integrates with the host genome. Vital to enhancing the rational design of LV vectors is the detailed understanding of the steps involving this infectivity. However, many aspects of these steps are not fully understood.

[009] Vector production on a laboratory or commercial scale requires the interaction of several biological inputs (e.g., plasmids, viral inoculum, auxiliary helper genes, and host cells) within a controlled cell culture environment (Aponte-Ubillus et ah, 2018). This process is currently carried out using mammalian cells (e.g., HEK293 and BHK cells) or insect cells (e.g., Sf9) genetically modified to express AAV proteins. For example, Sf9 cells are routinely used for commercial production of recombinant proteins, vaccines, biologies or gene delivery vehicles such as recombinant adeno-associated virus (rAAV) vectors. Expression of the capsid proteins VP1, VP2, and VP3 leads to the formation of viral capsids in the nucleolus (Samulski and Muzyczka, 2014). The expression of the non- structural proteins Rep78/68 and Rep52/40 triggers rAAV DNA replication and encapsidation of the generated single-stranded sequence (Balakrishnan and Jayandharan, 2014). In mammalian cells, the expression of auxiliary adenovirus or herpesvirus proteins is necessary to complement rAAV production and the identified helper genes participate as trans-activating agents of AAV promoters, or modifiers of the host cell milieu (Geoffroy and Salvetti, 2005).

SUMMARY OF THE INVENTION

[0010] The present disclosure relates to cell culture mediums and feed formulations used with mammalian cells for recombinant GT virus (rGTV) or rAAV production, as well as methods of generating AAV in host cells and host cell having vectors for rGTV or rAAV production in culture with the cell culture mediums. The inventors surprisingly discovered that the cell culture mediums and feed formulations of various embodiments improved rGTV or rAAV production by the cells (e.g., rAAV titer).

[0011] In various embodiments, the cell culture medium comprises a component, wherein the component, when cultured with a host cell, increases nicotinamide adenine dinucleotide biosynthesis or decreases nicotinamide adenine dinucleotide degradation within the host cell.

The component increases rGTV or rAAV production by a host cell comprising one or more vectors for rGTV or rAAV production relative to rGTV or rAAV production by the host cell comprising one or more vectors for rGTV or rAAV production, which is cultured with a cell culture medium without the component. In various embodiments, a method of generating rGTV or rAAV production comprises culturing a host cell comprising one or more vectors for rGTV or rAAV production with the cell culture medium comprising the component. In various embodiments host cells having vectors for rGTV or rAAV production are grown in culture with the cell culture medium having the component.

[0012] In various embodiments, a feed formulation comprises a component that, when cultured with a cell, increases nicotinamide adenine dinucleotide biosynthesis within the cell.

The formulation is added to a cell culture containing a cell culture medium and a host cell comprising one or more vectors for rGTV or rAAV production at a predetermined rate, such that the cell culture medium comprises a desired concentration of the component for a predetermined time. The concentration of the component increases the rGTV or rAAV production by the host cell relative to rGTV or rAAV production by the host cell comprising one or more vectors for rGTV or rAAV production, which is cultured with the cell culture medium without the desired concentration of the component. In various embodiments, a method of generating rGTV or rAAV production comprises the steps of culturing a host cell comprising one or more vectors for rGTV or rAAV production in a cell culture medium and adding the feed formulation comprising the component to the cell culture medium at a predetermined rate, such that the cell culture media comprises a desired concentration of the component for a predetermined time. The desired concentration of the component increases rGTV or rAAV production by the host cell relative to rGTV or rAAV production by a host cell comprising one or more vectors for rGTV or rAAV production, which is cultured with a cell culture medium without the concentration of the component. Various embodiments comprise host cells having vectors for rGTV or rAAV production in culture with the cell culture medium to which a feed formulation comprising a component that, when cultured with a cell, increases nicotinamide adenine dinucleotide biosynthesis within the cell, is added. With the host cells the formulation is added to the cell culture containing the cell culture medium at a predetermined rate, such that the cell culture medium comprises a desired concentration of the component for a predetermined time. The desired concentration of the component increases rGTV or rAAV production by the host cells relative to rGTV or rAAV production by a host cell comprising one or more vectors for rGTV or rAAV production, which is cultured with a cell culture medium without the concentration of the component.

[0013] In various embodiment, a cell culture medium comprises a nicotinamide concentration of 1 mM or more.

[0014] In various embodiments, methods of generating rGTV or rAAV are disclosed. The methods comprise the step of culturing a host cell comprising one or more vectors for rGTV or rAAV production in the cell culture medium of various embodiments.

[0015] In various embodiments, a feed formulation comprises nicotinamide. The feed formulation is added to a cell culture containing a host cell and a cell culture medium at a predetermined rate such that the cell culture media comprises a nicotinamide concentration of 1 mM or more for a predetermined time.

[0016] In various embodiments, methods of generating rGTV or rAAV are disclosed. The methods comprise the steps of culturing a host cell comprising one or more vectors for rGTV or rAAV production in a cell culture medium and adding the feed formulation of various embodiments to the cell culture medium at a predetermined rate such that the cell culture media comprises a nicotinamide concentration of 1 mM or more for a predetermined time.

[0017] In various embodiments, host cells having vectors for rGTV or rAAV production in culture with the cell culture medium to which nicotinamide at concentration of 1 mM or more for a predetermined time has been added are disclosed. Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to one of ordinary skill in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will become more fully understood from the detailed description given below and the accompanying drawings that are given by way of illustration only and are thus not limitative of the present invention.

[0019] Figure 1 illustrates the effect of the feed formulation of the present invention on Bba41 titers at a high cell density. HEK293 cells were cultured in media of choice and transfected with plasmids for generating rAAV.

[0020] Figure 2 illustrates the effect of the addition of a feed formulation of the present invention on rAAV9 titers at two different cell densities in Ambrl5 mini bioreactors. HEK293 cells were transfected with plasmids for generating rAAV.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will now be described with reference to the accompanying drawings.

[0022] Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word “about.” The first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property. It is also understood that disclosed ranges include the endpoints defining the range.

[0023] Unless indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0024] It is also to be understood that this disclosure is not limited to the specific embodiments and methods described below, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for describing particular embodiments and is not intended to be limiting in any way.

[0025] It must also be noted that, as used in the specification and the appended claims, the singular form "a," "an," and "the" comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.

[0026] The terms “or” and “and” can be used interchangeably and can be understood to mean “and/or.”

[0027] The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.

[0028] The phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0029] The phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0030] The terms “comprising,” “consisting of,” and “consisting essentially of’ can be alternatively used. When one of these three terms is used, the presently disclosed and claimed subject matter can include the use of either of the other two terms. [0031] Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

[0032] The present invention is directed to cell culture mediums and feed formulations for rGTV or rAAV production as well as host cells having vectors for rGTV or rAAV production in culture with the cell culture medium. Specifically, the present invention provides increased titers of rGTV or rAAV when compared to conventional techniques. In particular, the present invention provides increased titers of rGTV or rAAV in mammalian cells when compared to conventional techniques.

[0033] Cell Culture Medium and Feed Formulations

[0034] In various embodiments, a cell culture medium comprises a concentration of a component, where the component when cultured with a host cell increases nicotinamide adenine dinucleotide biosynthesis within the host cell. The concentration of the component increases rGTV or rAAV production by a host cell comprising one or more vectors for rGTV or rAAV production relative to rGTV or rAAV production by a host cell comprising one or more vectors for rGTV or rAAV production that is cultured with a cell culture medium without the concentration of the component. In various embodiments, a method of generating rGTV or rAAV production comprises the step of culturing a host cell comprising one or more vectors for rGTV or rAAV production with the cell culture medium comprising the concentration of component.

[0035] In various embodiments, a feed formulation comprises a component that when cultured with a cell increases nicotinamide adenine dinucleotide biosynthesis within the cell. The formulation is added to a cell culture containing a cell culture medium and a host cell comprising one or more vectors for rGTV or rAAV production at a predetermined rate such that the cell culture medium comprises a concentration of the component for a predetermined time. The concentration of the component increases rGTV or rAAV production by the host cell relative to rGTV or rAAV production by a host cell comprising one or more vectors for rGTV or rAAV production that is cultured with a cell culture medium without the concentration of the component. In various embodiments, a method of generating rGTV or rAAV production comprises the steps of culturing a host cell comprising one or more vectors for rGTV or rAAV production in a cell culture medium and adding the feed formulation comprising the component to the cell culture medium at a predetermined rate such that the cell culture media comprises a concentration of the component for a predetermined time. The concentration of the component increases rGTV or rAAV production by the host cell relative to rGTV or rAAV production by a host cell comprising one or more vectors for rGTV or rAAV production that is cultured with a cell culture medium without the concentration of the component.

[0036] In various embodiments, the component of the cell culture medium or feed formulation of different embodiments includes nicotinamide, niacin, nicotinamide mononucleotide, nicotinamide riboside, and combinations thereof. The component of various embodiments can also include components used in nicotinamide-adenine dinucleotide biosynthesis including intermediates or precursors such as nicotinate, quinolinate, nicotinamide mononucleotide, nicotinamide riboside, or niacin, as well as nicotinamide. The component of various embodiments can also be modified. For example, the component can be covalently modified such as with linkage to methyl groups, polyethylene glycol, or palmitoyl groups to increase cell permeability.

[0037] In various embodiments, a cell culture medium comprises a nicotinamide concentration of 1 millimolar (mM) or more.

[0038] In various embodiments, a feed formulation comprises nicotinamide. The feed formulation is added to a cell culture containing a host cell and a cell culture medium at a predetermined rate such that the cell culture media comprises a nicotinamide concentration of 1 mM or more for a predetermined time.

[0039] In various embodiments, the nicotinamide concentration is 1 mM to 500 mM, 1 mM to 250 mM, 1 mM to 100 mM, 5 mM to 100 mM, 5 mM to 25 mM, or 10 mM to 20 mM.

[0040] In various embodiments, the nicotinamide concentration is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM,

104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, 120 mM, 121 mM, 122 mM, 123 mM, 124 mM, 125 mM, 126 mM, 127 mM, 128 mM, 129 mM, 130 mM, 131 mM, 132 mM, 133 mM, 134 mM, 135 mM, 136 mM, 137 mM, 138 mM, 139 mM, 140 mM, 141 mM, 142 mM, 143 mM, 144 mM, 145 mM, 146 mM, 147 mM, 148 mM, 149 mM, 150 mM, 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, 156 mM, 157 mM, 158 mM, 159 mM, 160 mM, 161 mM, 162 mM, 163 mM, 164 mM, 165 mM, 166 mM, 167 mM, 168 mM, 169 mM, 170 mM, 171 mM, 172 mM, 173 mM, 174 mM, 175 mM, 176 mM, 177 mM, 178 mM, 179 mM, 180 mM, 181 mM, 182 mM, 183 mM, 184 mM, 185 mM, 186 mM, 187 mM, 188 mM, 189 mM, 190 mM, 191 mM, 192 mM, 193 mM, 194 mM, 195 mM, 196 mM, 197 mM, 198 mM, 199 mM, 200 mM, 201 mM, 202 mM, 203 mM, 204 mM, 205 mM, 206 mM, 207 mM, 208 mM, 209 mM, 210 mM, 211 mM, 212 mM, 213 mM, 214 mM, 215 mM, 216 mM, 217 mM, 218 mM, 219 mM, 220 mM, 221 mM, 222 mM, 223 mM, 224 mM, 225 mM, 226 mM, 227 mM, 228 mM, 229 mM, 230 mM, 231 mM, 232 mM, 233 mM, 234 mM, 235 mM, 236 mM, 237 mM, 238 mM, 239 mM, 240 mM, 241 mM, 242 mM, 243 mM, 244 mM, 245 mM, 246 mM, 247 mM, 248 mM, 249 mM, 250 mM, 251 mM, 252 mM, 253 mM, 254 mM, 255 mM, 256 mM, 257 mM, 258 mM, 259 mM, 260 mM, 261 mM, 262 mM, 263 mM, 264 mM, 265 mM, 266 mM, 267 mM, 268 mM, 269 mM, 270 mM, 271 mM, 272 mM, 273 mM, 274 mM, 275 mM, 276 mM, 277 mM, 278 mM, 279 mM, 280 mM, 281 mM, 282 mM, 283 mM, 284 mM, 285 mM, 286 mM, 287 mM, 288 mM, 289 mM, 290 mM, 291 mM, 292 mM, 293 mM, 294 mM, 295 mM, 296 mM, 297 mM, 298 mM, 299 mM, 300 mM, 301 mM, 302 mM, 303 mM, 304 mM, 305 mM, 306 mM, 307 mM, 308 mM, 309 mM, 310 mM, 311 mM, 312 mM, 313 mM, 314 mM, 315 mM, 316 mM, 317 mM, 318 mM, 319 mM, 320 mM, 321 mM, 322 mM, 323 mM, 324 mM, 325 mM, 326 mM, 327 mM, 328 mM, 329 mM, 330 mM, 331 mM, 332 mM, 333 mM, 334 mM, 335 mM, 336 mM, 337 mM, 338 mM, 339 mM, 340 mM, 341 mM, 342 mM, 343 mM, 344 mM, 345 mM, 346 mM, 347 mM, 348 mM, 349 mM, 350 mM, 351 mM, 352 mM, 353 mM, 354 mM, 355 mM, 356 mM, 357 mM, 358 mM, 359 mM, 360 mM, 361 mM, 362 mM, 363 mM, 364 mM, 365 mM, 366 mM, 367 mM, 368 mM, 369 mM, 370 mM, 371 mM, 372 mM, 373 mM, 374 mM, 375 mM, 376 mM, 377 mM, 378 mM, 379 mM, 380 mM, 381 mM, 382 mM, 383 mM, mM, 385 mM, 386 mM, 387 mM, 388 mM, 389 mM, 390 mM, 391 mM, 392 mM, 393 mM, mM, 395 mM, 396 mM, 397 mM, 398 mM, 399 mM, 400 mM, 401 mM, 402 mM, 403 mM, mM, 405 mM, 406 mM, 407 mM, 408 mM, 409 mM, 410 mM, 411 mM, 412 mM, 413 mM, mM, 415 mM, 416 mM, 417 mM, 418 mM, 419 mM, 420 mM, 421 mM, 422 mM, 423 mM, mM, 425 mM, 426 mM, 427 mM, 428 mM, 429 mM, 430 mM, 431 mM, 432 mM, 433 mM, mM, 435 mM, 436 mM, 437 mM, 438 mM, 439 mM, 440 mM, 441 mM, 442 mM, 443 mM, mM, 445 mM, 446 mM, 447 mM, 448 mM, 449 mM, 450 mM, 451 mM, 452 mM, 453 mM, mM, 455 mM, 456 mM, 457 mM, 458 mM, 459 mM, 460 mM, 461 mM, 462 mM, 463 mM, mM, 465 mM, 466 mM, 467 mM, 468 mM, 469 mM, 470 mM, 471 mM, 472 mM, 473 mM, mM, 475 mM, 476 mM, 477 mM, 478 mM, 479 mM, 480 mM, 481 mM, 482 mM, 483 mM, mM, 485 mM, 486 mM, 487 mM, 488 mM, 489 mM, 490 mM, 491 mM, 492 mM, 493 mM, mM, 495 mM, 496 mM, 497 mM, 498 mM, 499 mM, 500 mM, 501 mM, 502 mM, 503 mM, mM, 505 mM, 506 mM, 507 mM, 508 mM, 509 mM, 510 mM, 511 mM, 512 mM, 513 mM, mM, 515 mM, 516 mM, 517 mM, 518 mM, 519 mM, 520 mM, 521 mM, 522 mM, 523 mM, mM, 525 mM, 526 mM, 527 mM, 528 mM, 529 mM, 530 mM, 531 mM, 532 mM, 533 mM, mM, 535 mM, 536 mM, 537 mM, 538 mM, 539 mM, 540 mM, 541 mM, 542 mM, 543 mM, mM, 545 mM, 546 mM, 547 mM, 548 mM, 549 mM, 550 mM, 551 mM, 552 mM, 553 mM, mM, 555 mM, 556 mM, 557 mM, 558 mM, 559 mM, 560 mM, 561 mM, 562 mM, 563 mM, mM, 565 mM, 566 mM, 567 mM, 568 mM, 569 mM, 570 mM, 571 mM, 572 mM, 573 mM, mM, 575 mM, 576 mM, 577 mM, 578 mM, 579 mM, 580 mM, 581 mM, 582 mM, 583 mM, mM, 585 mM, 586 mM, 587 mM, 588 mM, 589 mM, 590 mM, 591 mM, 592 mM, 593 mM, mM, 595 mM, 596 mM, 597 mM, 598 mM, 599 mM, 600 mM, 601 mM, 602 mM, 603 mM, mM, 605 mM, 606 mM, 607 mM, 608 mM, 609 mM, 610 mM, 611 mM, 612 mM, 613 mM, mM, 615 mM, 616 mM, 617 mM, 618 mM, 619 mM, 620 mM, 621 mM, 622 mM, 623 mM, mM, 625 mM, 626 mM, 627 mM, 628 mM, 629 mM, 630 mM, 631 mM, 632 mM, 633 mM, mM, 635 mM, 636 mM, 637 mM, 638 mM, 639 mM, 640 mM, 641 mM, 642 mM, 643 mM, mM, 645 mM, 646 mM, 647 mM, 648 mM, 649 mM, 650 mM, 651 mM, 652 mM, 653 mM, mM, 655 mM, 656 mM, 657 mM, 658 mM, 659 mM, 660 mM, 661 mM, 662 mM, 663 mM, mM, 665 mM, 666 mM, 667 mM, 668 mM, 669 mM, 670 mM, 671 mM, 672 mM, 673 mM, mM, 675 mM, 676 mM, 677 mM, 678 mM, 679 mM, 680 mM, 681 mM, 682 mM, 683 mM, mM, 685 mM, 686 mM, 687 mM, 688 mM, 689 mM, 690 mM, 691 mM, 692 mM, 693 mM, mM, 695 mM, 696 mM, 697 mM, 698 mM, 699 mM, 700 mM, 701 mM, 702 mM, 703 mM, mM, 705 mM, 706 mM, 707 mM, 708 mM, 709 mM, 710 mM, 711 mM, 712 mM, 713 mM, mM, 715 mM, 716 mM, 717 mM, 718 mM, 719 mM, 720 mM, 721 mM, 722 mM, 723 mM, mM, 725 mM, 726 mM, 727 mM, 728 mM, 729 mM, 730 mM, 731 mM, 732 mM, 733 mM, mM, 735 mM, 736 mM, 737 mM, 738 mM, 739 mM, 740 mM, 741 mM, 742 mM, 743 mM, mM, 745 mM, 746 mM, 747 mM, 748 mM, 749 mM, 750 mM, 751 mM, 752 mM, 753 mM, mM, 755 mM, 756 mM, 757 mM, 758 mM, 759 mM, 760 mM, 761 mM, 762 mM, 763 mM, mM, 765 mM, 766 mM, 767 mM, 768 mM, 769 mM, 770 mM, 771 mM, 772 mM, 773 mM, mM, 775 mM, 776 mM, 777 mM, 778 mM, 779 mM, 780 mM, 781 mM, 782 mM, 783 mM, mM, 785 mM, 786 mM, 787 mM, 788 mM, 789 mM, 790 mM, 791 mM, 792 mM, 793 mM, mM, 795 mM, 796 mM, 797 mM, 798 mM, 799 mM, 800 mM, 801 mM, 802 mM, 803 mM, mM, 805 mM, 806 mM, 807 mM, 808 mM, 809 mM, 810 mM, 811 mM, 812 mM, 813 mM, mM, 815 mM, 816 mM, 817 mM, 818 mM, 819 mM, 820 mM, 821 mM, 822 mM, 823 mM, mM, 825 mM, 826 mM, 827 mM, 828 mM, 829 mM, 830 mM, 831 mM, 832 mM, 833 mM, mM, 835 mM, 836 mM, 837 mM, 838 mM, 839 mM, 840 mM, 841 mM, 842 mM, 843 mM, mM, 845 mM, 846 mM, 847 mM, 848 mM, 849 mM, 850 mM, 851 mM, 852 mM, 853 mM, mM, 855 mM, 856 mM, 857 mM, 858 mM, 859 mM, 860 mM, 861 mM, 862 mM, 863 mM, mM, 865 mM, 866 mM, 867 mM, 868 mM, 869 mM, 870 mM, 871 mM, 872 mM, 873 mM, mM, 875 mM, 876 mM, 877 mM, 878 mM, 879 mM, 880 mM, 881 mM, 882 mM, 883 mM, mM, 885 mM, 886 mM, 887 mM, 888 mM, 889 mM, 890 mM, 891 mM, 892 mM, 893 mM, mM, 895 mM, 896 mM, 897 mM, 898 mM, 899 mM, 900 mM, 901 mM, 902 mM, 903 mM, mM, 905 mM, 906 mM, 907 mM, 908 mM, 909 mM, 910 mM, 911 mM, 912 mM, 913 mM, mM, 915 mM, 916 mM, 917 mM, 918 mM, 919 mM, 920 mM, 921 mM, 922 mM, 923 mM, mM, 925 mM, 926 mM, 927 mM, 928 mM, 929 mM, 930 mM, 931 mM, 932 mM, 933 mM, mM, 935 mM, 936 mM, 937 mM, 938 mM, 939 mM, 940 mM, 941 mM, 942 mM, 943 mM, mM, 945 mM, 946 mM, 947 mM, 948 mM, 949 mM, 950 mM, 951 mM, 952 mM, 953 mM, mM, 955 mM, 956 mM, 957 mM, 958 mM, 959 mM, 960 mM, 961 mM, 962 mM, 963 mM, mM, 965 mM, 966 mM, 967 mM, 968 mM, 969 mM, 970 mM, 971 mM, 972 mM, 973 mM, mM, 975 mM, 976 mM, 977 mM, 978 mM, 979 mM, 980 mM, 981 mM, 982 mM, 983 mM, mM, 985 mM, 986 mM, 987 mM, 988 mM, 989 mM, 990 mM, 991 mM, 992 mM, 993 mM, 994 mM, 995 mM, 996 mM, 997 mM, 998 mM, 999 mM, or 1000 mM. In other embodiments, the nicotinamide concentration is a range between any two concentrations provided above.

[0041] In various embodiments, the cell culture medium of various embodiments and the feed formulation of various embodiments comprise one or more concentration(s) of carnitine, serine, arginine, taurine, ornithine, vitamins, fatty acids, and free cholesterol. In other embodiments, the cell culture medium of various embodiments comprises one or more concentration(s) of 1 mM or more of carnitine, 1 mM or more of serine, 1 mM or more of arginine, 1 mM or more of taurine,

1 mM or more of ornithine, 0.1 mg/L or more of vitamins, fatty acids, and free cholesterol. The cell culture medium of various embodiments and the feed formulation of various embodiments can also include other types of nonessential amino acids such as glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine. The cell culture medium of various embodiments and the feed formulation of various embodiments can also include other amino acids such as arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. It is noted that the nonessential amino acids and other amino acids include salts and enantiomers.

[0042] In various embodiments, the concentration of carnitine, serine, arginine, taurine, or ornithine is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, 120 mM, 121 mM, 122 mM, 123 mM, 124 mM, 125 mM, 126 mM, 127 mM, 128 mM, 129 mM, 130 mM, 131 mM, 132 mM, 133 mM, 134 mM, 135 mM, 136 mM, 137 mM, 138 mM, 139 mM, 140 mM, 141 mM, 142 mM, 143 mM, 144 mM, 145 mM, 146 mM, 147 mM, 148 mM, 149 mM, 150 mM, 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, 156 mM, 157 mM, 158 mM, 159 mM, 160 mM, 161 mM, 162 mM, 163 mM, 164 mM, 165 mM, 166 mM, 167 mM, 168 mM, 169 mM, 170 mM, 171 mM, 172 mM, 173 mM, 174 mM, 175 mM, 176 mM, 177 mM, 178 mM, 179 mM, 180 mM, 181 mM, 182 mM, 183 mM, 184 mM, 185 mM, 186 mM, 187 mM, 188 mM, 189 mM, 190 mM, 191 mM, 192 mM, 193 mM, 194 mM, 195 mM, 196 mM, 197 mM, 198 mM, 199 mM, 200 mM, 201 mM, 202 mM, 203 mM, 204 mM, 205 mM, 206 mM, 207 mM, 208 mM, 209 mM, 210 mM, 211 mM, 212 mM, 213 mM, 214 mM, 215 mM, 216 mM, 217 mM, 218 mM, 219 mM, 220 mM, 221 mM, 222 mM, 223 mM, 224 mM, 225 mM, 226 mM, 227 mM, 228 mM, 229 mM, 230 mM, 231 mM, 232 mM, 233 mM, 234 mM, 235 mM, 236 mM, 237 mM, 238 mM, 239 mM, 240 mM, 241 mM, 242 mM, 243 mM, 244 mM, 245 mM, 246 mM, 247 mM, 248 mM, 249 mM, 250 mM, 251 mM, 252 mM, 253 mM, 254 mM, 255 mM, 256 mM, 257 mM, 258 mM, 259 mM, 260 mM, 261 mM, 262 mM, 263 mM, 264 mM, 265 mM, 266 mM, 267 mM, 268 mM, 269 mM, 270 mM, 271 mM, 272 mM, 273 mM, 274 mM, 275 mM, 276 mM, 277 mM, 278 mM, 279 mM, 280 mM, 281 mM, 282 mM, 283 mM, 284 mM, 285 mM, 286 mM, 287 mM, 288 mM, 289 mM, 290 mM, 291 mM, 292 mM, 293 mM, 294 mM, 295 mM, 296 mM, 297 mM, 298 mM, 299 mM, 300 mM, 301 mM, 302 mM, 303 mM, 304 mM, 305 mM, 306 mM, 307 mM, 308 mM, 309 mM, 310 mM, 311 mM, 312 mM, 313 mM, 314 mM, 315 mM, 316 mM, 317 mM, 318 mM, 319 mM, 320 mM, 321 mM, 322 mM, 323 mM, 324 mM, 325 mM, 326 mM, 327 mM, 328 mM, 329 mM, 330 mM, 331 mM, 332 mM, 333 mM, 334 mM, 335 mM, 336 mM, 337 mM, 338 mM, 339 mM, 340 mM, 341 mM, 342 mM, 343 mM, 344 mM, 345 mM, 346 mM, 347 mM, 348 mM, 349 mM, 350 mM, 351 mM, 352 mM, 353 mM, 354 mM, 355 mM, 356 mM, 357 mM, 358 mM, 359 mM, 360 mM, 361 mM, 362 mM, 363 mM, 364 mM, 365 mM, 366 mM, 367 mM, 368 mM, 369 mM, 370 mM, 371 mM, 372 mM, 373 mM, 374 mM, 375 mM, 376 mM, 377 mM, 378 mM, 379 mM, 380 mM, 381 mM, 382 mM, 383 mM, 384 mM, 385 mM, 386 mM, 387 mM, 388 mM, 389 mM, 390 mM, 391 mM, 392 mM, 393 mM, 394 mM, 395 mM, 396 mM, 397 mM, 398 mM, 399 mM, 400 mM, 401 mM, 402 mM, 403 mM, 404 mM, 405 mM, 406 mM, 407 mM, 408 mM, 409 mM, 410 mM, 411 mM, 412 mM, 413 mM, 414 mM, 415 mM, 416 mM, 417 mM, 418 mM, 419 mM, 420 mM, 421 mM, 422 mM, 423 mM, 424 mM, 425 mM, 426 mM, 427 mM, 428 mM, 429 mM, 430 mM, 431 mM, 432 mM, 433 mM, 434 mM, 435 mM, 436 mM, 437 mM, 438 mM, 439 mM, 440 mM, 441 mM, 442 mM, 443 mM, 444 mM, 445 mM, 446 mM, 447 mM, 448 mM, 449 mM, 450 mM, 451 mM, 452 mM, 453 mM, 454 mM, 455 mM, 456 mM, 457 mM, 458 mM, 459 mM, 460 mM, 461 mM, 462 mM, 463 mM, 464 mM, 465 mM, 466 mM, 467 mM, 468 mM, 469 mM, 470 mM, 471 mM, 472 mM, 473 mM, 474 mM, 475 mM, 476 mM, 477 mM, 478 mM, 479 mM, 480 mM, 481 mM, 482 mM, 483 mM, 484 mM, 485 mM, 486 mM, 487 mM, 488 mM, 489 mM, 490 mM, 491 mM, 492 mM, 493 mM, 494 mM, 495 mM, 496 mM, 497 mM, 498 mM, 499 mM, 500 mM, 501 mM, 502 mM, 503 mM, 504 mM, 505 mM, 506 mM, 507 mM, 508 mM, 509 mM, 510 mM, 511 mM, 512 mM, 513 mM, 514 mM, 515 mM, 516 mM, 517 mM, 518 mM, 519 mM, 520 mM, 521 mM, 522 mM, 523 mM, 524 mM, 525 mM, 526 mM, 527 mM, 528 mM, 529 mM, 530 mM, 531 mM, 532 mM, 533 mM, 534 mM, 535 mM, 536 mM, 537 mM, 538 mM, 539 mM, 540 mM, 541 mM, 542 mM, 543 mM, 544 mM, 545 mM, 546 mM, 547 mM, 548 mM, 549 mM, 550 mM, 551 mM, 552 mM, 553 mM, 554 mM, 555 mM, 556 mM, 557 mM, 558 mM, 559 mM, 560 mM, 561 mM, 562 mM, 563 mM, 564 mM, 565 mM, 566 mM, 567 mM, 568 mM, 569 mM, 570 mM, 571 mM, 572 mM, 573 mM, 574 mM, 575 mM, 576 mM, 577 mM, 578 mM, 579 mM, 580 mM, 581 mM, 582 mM, 583 mM, 584 mM, 585 mM, 586 mM, 587 mM, 588 mM, 589 mM, 590 mM, 591 mM, 592 mM, 593 mM, 594 mM, 595 mM, 596 mM, 597 mM, 598 mM, 599 mM, 600 mM, 601 mM, 602 mM, 603 mM, 604 mM, 605 mM, 606 mM, 607 mM, 608 mM, 609 mM, 610 mM, 611 mM, 612 mM, 613 mM, 614 mM, 615 mM, 616 mM, 617 mM, 618 mM, 619 mM, 620 mM, 621 mM, 622 mM, 623 mM, 624 mM, 625 mM, 626 mM, 627 mM, 628 mM, 629 mM, 630 mM, 631 mM, 632 mM, 633 mM, 634 mM, 635 mM, 636 mM, 637 mM, 638 mM, 639 mM, 640 mM, 641 mM, 642 mM, 643 mM, 644 mM, 645 mM, 646 mM, 647 mM, 648 mM, 649 mM, 650 mM, 651 mM, 652 mM, 653 mM, 654 mM, 655 mM, 656 mM, 657 mM, 658 mM, 659 mM, 660 mM, 661 mM, 662 mM, 663 mM, 664 mM, 665 mM, 666 mM, 667 mM, 668 mM, 669 mM, 670 mM, 671 mM, 672 mM, 673 mM, 674 mM, 675 mM, 676 mM, 677 mM, 678 mM, 679 mM, 680 mM, 681 mM, 682 mM, 683 mM, 684 mM, 685 mM, 686 mM, 687 mM, 688 mM, 689 mM, 690 mM, 691 mM, 692 mM, 693 mM, 694 mM, 695 mM, 696 mM, 697 mM, 698 mM, 699 mM, 700 mM, 701 mM, 702 mM, 703 mM, 704 mM, 705 mM, 706 mM, 707 mM, 708 mM, 709 mM, 710 mM, 711 mM, 712 mM, 713 mM, 714 mM, 715 mM, 716 mM, 717 mM, 718 mM, 719 mM, 720 mM, 721 mM, 722 mM, 723 mM, 724 mM, 725 mM, 726 mM, 727 mM, 728 mM, 729 mM, 730 mM, 731 mM, 732 mM, 733 mM, 734 mM, 735 mM, 736 mM, 737 mM, 738 mM, 739 mM, 740 mM, 741 mM, 742 mM, 743 mM, 744 mM, 745 mM, 746 mM, 747 mM, 748 mM, 749 mM, 750 mM, 751 mM, 752 mM, 753 mM, 754 mM, 755 mM, 756 mM, 757 mM, 758 mM, 759 mM, 760 mM, 761 mM, 762 mM, 763 mM, 764 mM, 765 mM, 766 mM, 767 mM, 768 mM, 769 mM, 770 mM, 771 mM, 772 mM, 773 mM, 774 mM, 775 mM, 776 mM, 777 mM, 778 mM, 779 mM, 780 mM, 781 mM, 782 mM, 783 mM, 784 mM, 785 mM, 786 mM, 787 mM, 788 mM, 789 mM, 790 mM, 791 mM, 792 mM, 793 mM, 794 mM, 795 mM, 796 mM, 797 mM, 798 mM, 799 mM, 800 mM, 801 mM, 802 mM, 803 mM, 804 mM, 805 mM, 806 mM, 807 mM, 808 mM, 809 mM, 810 mM, 811 mM, 812 mM, 813 mM, 814 mM, 815 mM, 816 mM, 817 mM, 818 mM, 819 mM, 820 mM, 821 mM, 822 mM, 823 mM, 824 mM, 825 mM, 826 mM, 827 mM, 828 mM, 829 mM, 830 mM, 831 mM, 832 mM, 833 mM, 834 mM, 835 mM, 836 mM, 837 mM, 838 mM, 839 mM, 840 mM, 841 mM, 842 mM, 843 mM, 844 mM, 845 mM, 846 mM, 847 mM, 848 mM, 849 mM, 850 mM, 851 mM, 852 mM, 853 mM, 854 mM, 855 mM, 856 mM, 857 mM, 858 mM, 859 mM, 860 mM, 861 mM, 862 mM, 863 mM, 864 mM, 865 mM, 866 mM, 867 mM, 868 mM, 869 mM, 870 mM, 871 mM, 872 mM, 873 mM, 874 mM, 875 mM, 876 mM, 877 mM, 878 mM, 879 mM, 880 mM, 881 mM, 882 mM, 883 mM, 884 mM, 885 mM, 886 mM, 887 mM, 888 mM, 889 mM, 890 mM, 891 mM, 892 mM, 893 mM, 894 mM, 895 mM, 896 mM, 897 mM, 898 mM, 899 mM, 900 mM, 901 mM, 902 mM, 903 mM, 904 mM, 905 mM, 906 mM, 907 mM, 908 mM, 909 mM, 910 mM, 911 mM, 912 mM, 913 mM, 914 mM, 915 mM, 916 mM, 917 mM, 918 mM, 919 mM, 920 mM, 921 mM, 922 mM, 923 mM, 924 mM, 925 mM, 926 mM, 927 mM, 928 mM, 929 mM, 930 mM, 931 mM, 932 mM, 933 mM, 934 mM, 935 mM, 936 mM, 937 mM, 938 mM, 939 mM, 940 mM, 941 mM, 942 mM, 943 mM, 944 mM, 945 mM, 946 mM, 947 mM, 948 mM, 949 mM, 950 mM, 951 mM, 952 mM, 953 mM, 954 mM, 955 mM, 956 mM, 957 mM, 958 mM, 959 mM, 960 mM, 961 mM, 962 mM, 963 mM, 964 mM, 965 mM, 966 mM, 967 mM, 968 mM, 969 mM, 970 mM, 971 mM, 972 mM, 973 mM, 974 mM, 975 mM, 976 mM, 977 mM, 978 mM, 979 mM, 980 mM, 981 mM, 982 mM, 983 mM, 984 mM, 985 mM, 986 mM, 987 mM, 988 mM, 989 mM, 990 mM, 991 mM, 992 mM, 993 mM, 994 mM, 995 mM, 996 mM, 997 mM, 998 mM, 999 mM, or

1000 mM. In other embodiments, the concentration of carnitine, serine, arginine, taurine, or ornithine is a range between any two concentrations provided above.

[0043] It is noted that cell culture medium of various embodiments and feed formulation of various embodiments includes variations, different versions, and modified forms of carnitine, serine, arginine, taurine, or ornithine, such as salts or enantiomers. For example, carnitine includes a carnitine inner salt. In another example, ornithine includes L-Ornithine L-Aspartate.

[0044] In various embodiments, the concentration of vitamins is 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.4 mg/L, 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1 mg/L, 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L, 60 mg/L, 70 mg/L, 80 mg/L, 90 mg/L, 100 mg/L. In other embodiments, the concentration of vitamins is a range between any two concentrations provided above.

[0045] In various embodiments, vitamins include but are not limited to one or more of choline chloride, D-calcium pantothenate, folic acid, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, i-inositol, and sodium chloride.

[0046] The fatty acids of various embodiments include fatty acids that are important for cell culture. For example, fatty acids of the n-3, n-6 and n-9 families are important supplements for cell culture systems. Linoleic acid is a n-6 unsaturated fatty acid that is not synthesized by mammalian cells and can be provided as a nutrient in cell culture. Linoleic acid (18:2, n-6) is also precursor to a number of other fatty acids, such as arachidonic acid (20:4, n-6), a PUFA with four double bonds that is a precursor of some prostaglandins. Linolenic (n-3) is a precursor to fatty acids, such as the docosahexaenoic acids (22:6, n-3), a highly unsaturated PUFA with six double bonds. Both docosahexaenoic acid and arachidonic acid are also other fatty acids that can be added.

[0047] The cholesterol of various embodiments includes but is not limited to cholesterols that improve growth and protein production of cells in culture. One example is a synthetic cholesterol (C27H46O; CAS number: 57-88-5). Other types of cholesterol are disclosed in US2014/0363845, which is incorporated in its entirety by reference.

[0048] In various embodiments, the cell culture medium of various embodiments or feed formulation of various embodiments comprise a Poly(ADP-Ribose) Polymerase 1 (PARP1) inhibitor. Examples of PARP1 inhibitors include 1,5-dihydroisoquinoline, 3,4-dihydro-5-methyl- l-[2H]-isoquinolinone, 8-hydroxy-2-methylquinazolin- 4-[3H]-one, or 3-aminobenzamide.

[0049] In various embodiments, methods of supplementing cell culture mediums of various embodiments in culture with feed formulations of various embodiments are disclosed. The feed formulations of various embodiments are added to the cell culture mediums of various embodiments at pre-determined rates for predetermined times such that cell culture mediums of various embodiments have a nicotinamide concentration of 1 mM or more for a pre-determined time. [0050] In various embodiments, the predetermined time for which the cell culture mediums of various embodiments have a nicotinamide concentration of 1 mM or more is at least 1 hour, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, or at least 96 hours or at any time interval within 1 to 240 hours.

[0051] In various embodiments, the predetermined time for which the cell culture mediums of various embodiments have a nicotinamide concentration of 1 mM or more is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, 96 hours, 97 hours, 98 hours, 99 hours, 100 hours, 101 hours, 102 hours, 103 hours, 104 hours, 105 hours, 106 hours, 107 hours, 108 hours, 109 hours, 110 hours, 111 hours, 112 hours, 113 hours, 114 hours, 115 hours, 116 hours, 117 hours, 118 hours, 119 hours, 120 hours, 121 hours, 122 hours, 123 hours, 124 hours, 125 hours, 126 hours, 127 hours, 128 hours, 129 hours, 130 hours, 131 hours, 132 hours, 133 hours, 134 hours, 135 hours, 136 hours, 137 hours, 138 hours, 139 hours, 140 hours, 141 hours, 142 hours, 143 hours, 144 hours, 145 hours, 146 hours, 147 hours, 148 hours, 149 hours, 150 hours, 151 hours, 152 hours, 153 hours, 154 hours, 155 hours, 156 hours, 157 hours, 158 hours, 159 hours, 160 hours, 161 hours, 162 hours, 163 hours, 164 hours, 165 hours, 166 hours, 167 hours, 168 hours, 169 hours, 170 hours, 171 hours, 172 hours, 173 hours, 174 hours, 175 hours, 176 hours, 177 hours, 178 hours, 179 hours, 180 hours, 181 hours, 182 hours, 183 hours, 184 hours, 185 hours, 186 hours, 187 hours, 188 hours, 189 hours, 190 hours, 191 hours, 192 hours, 193 hours, 194 hours, 195 hours, 196 hours, 197 hours, 198 hours, 199 hours, 200 hours, 201 hours, 202 hours, 203 hours, 204 hours, 205 hours, 206 hours, 207 hours, 208 hours, 209 hours, 210 hours, 211 hours, 212 hours, 213 hours, 214 hours, 215 hours, 216 hours, 217 hours, 218 hours, 219 hours, 220 hours, 221 hours, 222 hours, 223 hours, 224 hours, 225 hours, 226 hours, 227 hours, 228 hours, 229 hours, 230 hours, 231 hours, 232 hours, 233 hours, 234 hours,

235 hours, 236 hours, 237 hours, 238 hours, 239 hours, or 240 hours. In other embodiments, the predetermined time for which the cell culture mediums of various embodiments have a nicotinamide concentration of 1 mM or more is a range between any two times provided above.

[0052] In further embodiments, the addition of the feed formulation of various embodiments is added to the cell culture of various embodiments such that the cell culture of various embodiments also has concentrations of other elements (e.g., carnitine, serine, arginine, taurine, ornithine, vitamins, fatty acids, and free cholesterol) for a predetermined time. Example concentrations of the other elements are provided above. The predetermined time of various embodiments is at least 1 hour, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, or at least 96 hours. In various embodiments, the predetermined time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours,

26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours,

45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours,

64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours,

83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, 96 hours, 97 hours, 98 hours, 99 hours, 100 hours, 101 hours, 102 hours, 103 hours, 104 hours, 105 hours, 106 hours, 107 hours, 108 hours, 109 hours,

110 hours, 111 hours, 112 hours, 113 hours, 114 hours, 115 hours, 116 hours, 117 hours, 118 hours, 119 hours, 120 hours, 121 hours, 122 hours, 123 hours, 124 hours, 125 hours, 126 hours,

127 hours, 128 hours, 129 hours, 130 hours, 131 hours, 132 hours, 133 hours, 134 hours, 135 hours, 136 hours, 137 hours, 138 hours, 139 hours, 140 hours, 141 hours, 142 hours, 143 hours,

144 hours, 145 hours, 146 hours, 147 hours, 148 hours, 149 hours, 150 hours, 151 hours, 152 hours, 153 hours, 154 hours, 155 hours, 156 hours, 157 hours, 158 hours, 159 hours, 160 hours,

161 hours, 162 hours, 163 hours, 164 hours, 165 hours, 166 hours, 167 hours, 168 hours, 169 hours, 170 hours, 171 hours, 172 hours, 173 hours, 174 hours, 175 hours, 176 hours, 177 hours, 178 hours, 179 hours, 180 hours, 181 hours, 182 hours, 183 hours, 184 hours, 185 hours, 186 hours, 187 hours, 188 hours, 189 hours, 190 hours, 191 hours, 192 hours, 193 hours, 194 hours, 195 hours, 196 hours, 197 hours, 198 hours, 199 hours, 200 hours, 201 hours, 202 hours, 203 hours, 204 hours, 205 hours, 206 hours, 207 hours, 208 hours, 209 hours, 210 hours, 211 hours, 212 hours, 213 hours, 214 hours, 215 hours, 216 hours, 217 hours, 218 hours, 219 hours, 220 hours, 221 hours, 222 hours, 223 hours, 224 hours, 225 hours, 226 hours, 227 hours, 228 hours, 229 hours, 230 hours, 231 hours, 232 hours, 233 hours, 234 hours, 235 hours, 236 hours, 237 hours, 238 hours, 239 hours, or 240 hours. In other embodiments, the predetermined time is a range between any two times provided above.

[0053] Table 1 below provides one example of a feed formulation of the present invention.

Table 1 Composition of feed for rGTV or rAAV production

[0054] Table 2 below provides another example of a feed formulation of the present invention. Table 2 Composition of feed for rGTV or rAAV production [0055] Recombinant Viruses used for Gene Therapy and Adeno-Associated Virus [0056] The term “gene therapy” refers to the correction of defective genes by introducing normal genes or therapeutic genes into target cells that require gene therapy, or the prevention or treatment of genetic defects through genetic modification of cells by adding new functions to the cells. To this end, viruses such as retrovirus, lentivirus, adenovirus, adeno associated virus, and herpes simplex virus can be used as a vehicle for gene therapy.

[0057] The therapeutically effective rAAV particles include rAAV particles disclosed in US 9,504,762, WO 2019/222136, and US 2019/0376081, the disclosures of which are hereby incorporated by reference.

[0058] “AAV” is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus having a genome encapsidated by a capsid. There are currently thirteen serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228; and Bems, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to inverted terminal repeats (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0059] An “AAV viral particle” as used herein refers to an infectious viral particle composed of at least one AAV capsid protein and an encapsidated AAV genome. “Recombinant AAV” or “rAAV”, “rAAV virion” or “rAAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one capsid or Cap protein and an encapsidated rAAV vector genome as described herein. Thus, production of AAV vector particles includes production of an rAAV vector genome. The rAAV viral particle of different embodiments include AAV particles and rAAV particles disclosed in US 9,504,762, WO 2019/222136, and US 2019/0376081, the disclosures of which are hereby incorporated by reference.

[0060] “Capsid” refers to the structure in which the rAAV vector is packaged. The capsid includes VP1 proteins or VP3 proteins, but more typically, all three of VP1, VP2, and VP3 proteins, as found in native AAV. The sequence of the capsid proteins determines the serotype of the rAAV virions. rAAV virions include those derived from a number of AAV serotypes, including AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV13, Bba21, Bba26, Bba27, Bba29, Bba30, Bba31, Bba32, Bba33, Bba34, Bba35, Bba36, Bba37, Bba38, Bba41, Bba42, Bba43, Bba44, Bcel4, Bcel5, Bcel6, Bcel7, Bcel8, Bce20, Bce35, Bce36, Bce39, Bce40, Bce41, Bce42, Bce43, Bce44, Bce45, Bce46, Bey20, Bey22, Bey23, Bma42, Bma43, Bpol, Bpo2, Bpo3, Bpo4, Bpo6, Bpo8, Bpol3, Bpol8, Bpo20, Bpo23, Bpo24, Bpo27, Bpo28, Bpo29, Bpo33, Bpo35, Bpo36, Bpo37, Brh26, Brh27, Brh28, Brh29, Brh30, Brh31, Brh32, Brh33, Bfml7, Bfml8, Bfm20, Bfm21, Bfm24, Bfm25, Bfm27, Bfm32, Bfm33, Bfm34, Bfm35, AAV-rhlO, AAV-rh39, AAV-rh43, AAVanc80L65, or any variants thereof (see, e.g., U.S. Patent No. 8,318,480 for its disclosure of non-natural mixed serotypes). Exemplary capsids are also provided in International Application No. WO 2018/022608 and WO 2019/222136, which are each incorporated herein in their entirety. The capsid proteins can also be variants of natural VPl, VP2 and VP3, including mutated, chimeric or shuffled proteins. The capsid proteins can be those of rh.lO or other subtype within the various clades of AAV; various clades and subtypes are disclosed, for example, in U.S. Patent No. 7,906,111. In various embodiments, the capsid of the AAV viral particle has an acetylated or unacetylated VPl, VP2, or VP3 protein with an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a portion of an amino acid sequence from AAV-1 (NCBI Reference Sequence No. NP_049542.1), AAV-2 (NCBI Reference Sequence No. YP_680426.1), AAV-3 (NCBI Reference Sequence No. NP_043941.1), AAV-3B (Genbank Accession No. AAB95452.1), AAV-4 (NCBI Reference Sequence No. NP_044927.1), AAV- 5 (NCBI Reference Sequence No. YP_068409.1 or SEQ ID NO: 229), AAV-6 (Genbank Accession No. AAB95450.1), AAV-7 (NCBI Reference Sequence No. YP_077178.1), AAV-8 (NCBI Reference Sequence No. YP_077179.1), AAV-9 (Genbank Accession No. AAS99264.1), AAV-10 (Genbank Accession No. AAT46337.1), AAV-11 (Genbank Accession No. AAT46339.1), AAV-12 (Genbank Accession No. ABI16639.1), AAV-13 (Genbank Accession No. ABZ10812.1), or any amino acid sequence disclosed in WO 2018/022608 and WO 2019/222136. Construction and use of AAV proteins of different serotypes are discussed in Chao et al., Mol. Ther. 2:619-623, 2000; Davidson et al., PNAS 97:3428-3432, 2000; Xiao et al., J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol. 74:1524-1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, 2001.

[0061] As used herein, an “AAV vector genome”, “vector genome”, or “rAAV vector genome” refers to single-stranded nucleic acids. An rAAV viral particle has an rAAV vector genome encapsidated within a capsid. The rAAV vector genome has an AAV 5' inverted terminal repeat (ITR) sequence and an AAV 3' ITR flanking a protein-coding sequence (preferably a functional therapeutic protein-encoding sequence; e.g., FVIII, FIX, and PAH) operably linked to transcription regulatory elements that are heterologous to the AAV viral genome, i.e., one or more promoters and/or enhancers and, optionally, a polyadenylation sequence and/or one or more introns inserted in the regulatory elements or between the regulatory elements and the protein-coding sequence or between exons of the protein-coding sequence. rAAV vector genome refers to nucleic acids that are present in the rAAV virus particle and can be either the sense strand or the anti-sense strand of the nucleic acid sequences disclosed herein. The size of such single-stranded nucleic acids is provided in bases. The terms “inverted terminal repeat” and “ITR” as used herein refers to the art-recognized regions found at the 5' and 3' termini of the rAAV genome which function in cis as origins of viral DNA replication and as packaging signals for the viral genome. AAV ITRs, together with the Rep proteins, provide for efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a host cell genome. Sequences of certain AAV-associated ITRs are disclosed by Yan etal. , J. Virol. 79(l):364-379 (2005). ITRs are also found in a “flip” or “flop” configuration in which the sequence between the AA’ inverted repeats (that form the arms of the hairpin) are present in the reverse complement (Wilmott, Patrick, et al. Human gene therapy methods 30.6 (2019): 206-213). Construction and use of AAV vectors genomes of different serotypes are discussed in Chao et al., Mol. Ther. 2:619-623, 2000; Davidson et al., PNAS 97:3428-3432, 2000; Xiao et al., J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol. 74:1524- 1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; and Auricchio et al., Hum. Molec.

Genet. 10:3075-3081, 2001. Because of wide construct availability and extensive characterization, illustrative AAV vector genomes disclosed below are derived from serotype 2. [0062] As an example, a "therapeutic rAAV”, refers to an rAAV virion, rAAV viral particle, rAAV vector particle, or rAAV that comprises a heterologous polynucleotide that encodes a therapeutic protein, which can be used to replace or supplement the protein in vivo. The "therapeutic protein" is a polypeptide, whichhas a biological activity that replaces or compensates for the loss or reduction of activity of a corresponding endogenous protein. For example, a functional phenylalanine hydroxylase (PAH) is a therapeutic protein for phenylketonuria (PKU). Thus, for example recombinant AAV PAH virus can be used for a medicament for the treatment of a subject suffering from PKU. The medicament may be administered by intravenous (IV) administration and the administration of the medicament results in expression of PAH protein in the subject at levels sufficient to alter the neurotransmitter metabolite or neurotransmitter levels in the subject. Optionally, the medicament may also comprise a prophylactic and/or therapeutic corticosteroid for the prevention and/or treatment of any hepatotoxicity associated with administration of the rAAV PAH vector. The medicament comprising a prophylactic or therapeutic corticosteroid treatment may comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg/day of the corticosteroid. The medicament comprising a prophylactic or therapeutic corticosteroid may be administered over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more. The PKU therapy may optionally also include tyrosine supplements.

[0063] The transgene incorporated into the AAV capsid is not limited and may be any heterologous gene of therapeutic interest. The transgene is a nucleic acid sequence, which is heterologous to the AAV ITR sequences flanking the transgene, and which encodes a polypeptide, protein, or other product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a host cell.

[0064] The composition of the transgene sequence will depend upon the use to which the resulting vector will be put. For example, one type of transgene sequence includes a reporter sequence, which, upon expression, produces a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding b-lactamase, b-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.

[0065] These reporter coding sequences, when associated with regulatory elements, which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of cells infected by rAAV encoding the signal is detected by assays for beta-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be detected by instruments measuring fluorescence or luminescence.

[0066] However, the transgene is typically a non-marker sequence encoding a product which is useful in biology and medicine, such as proteins, peptides, RNA, enzymes, dominant negative mutants, or catalytic RNAs. Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNAs, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNAs. One example of a useful RNA sequence is a sequence which inhibits or extinguishes expression of a targeted nucleic acid sequence in the treated subject. Typically, suitable target sequences include oncologic targets and viral diseases. The transgene may be used to correct or ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed. A preferred type of transgene sequence encodes a therapeutic protein or polypeptide which is expressed in an infected cell. The vector may further include multiple transgenes, e.g., to correct or ameliorate a gene defect caused by a multi-subunit protein. In certain situations, a different transgene may be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large, e.g., for an immunoglobulin, the platelet-derived growth factor, or a dystrophin protein. In order for the cell to produce the multi-subunit protein, a cell is infected with the recombinant virus containing each of the different subunits. Alternatively, different subunits of a protein may be encoded by the same transgene. In this case, a single transgene includes the DNA encoding each of the subunits, with the DNA for each subunit separated by an internal ribozyme entry site (IRES). This may be the case when the size of the DNA encoding each of the subunits is small, e.g., the total size of the DNA encoding the subunits and the IRES is less than five kilobases. As an alternative to an IRES, the coding sequences may be separated by sequences encoding a 2A peptide, which self-cleaves in a post-translational event. See, e.g., Donnelly et al, ./. Gen. Virol ., 78(Pt 1): 13-21 (January 1997); Furler, et al, Gene Ther., 8(1 l):864-873 (June 2001); Klump et al, Gene Ther., 8(10):8 11- 817 (May 2001). A 2A peptide is significantly smaller than an IRES, making it well suited for use when space is a limiting factor. More often, when the transgene is large, consists of multi- subunits, or two transgenes are co-delivered, rAAV carrying the desired transgene(s) or subunits are co administered to allow them to concatamerize in vivo to form a single vector genome. In such an embodiment, a first AAV may carry an expression cassette which expresses a single transgene and a second AAV may carry an expression cassette which expresses a different transgene for co expression in the host cell. However, the selected transgene may encode any biologically active product or other product, e.g., a product desirable for study.

[0067] Suitable transgenes may be readily selected by one of ordinary skill in the art. The selection of the transgene is not considered to be a limitation of this invention. The transgene may be a heterologous protein, and this heterologous protein may be a therapeutic protein. Exemplary therapeutic proteins include, but are not limited to, blood factors, such as b-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony stimulating factors (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor- 9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a.), transforming growth factor beta (TGF-.b.), and the like; soluble receptors, such as soluble TNF-a. receptors, soluble VEGF receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), soluble g/d T cell receptors, ligand-binding fragments of a soluble receptor, and the like; enzymes, such as a- glucosidase, imiglucarase, b-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP-10, monokine induced by interferon-gamma (Mig), Groa/IL-8, RANTES, MIP-la, MIR- lb., MCP-1, PF-4, and the like; angiogenic agents, such as vascular endothelial growth factors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), glioma-derived growth factor, angiogenin, angiogenin-2; and the like; anti-angiogenic agents, such as a soluble VEGF receptor; protein vaccine; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); tissue factors, luteinizing hormone; macrophage activating factors; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); tissue inhibitors of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IF-1 receptor antagonists; and the like. Some other non-limiting examples of protein of interest include ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophins 3 and 4/5 (NT-3 and 4/5); glial cell derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia related clotting proteins, such as Factor VIII, Factor IX, Factor X; dystrophin, mini-dystrophin, or microdystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporter (e.g., GFUT2), aldolase A, b-enolase, and glycogen synthase; lysosomal enzymes (e.g., beta-N- acetylhexosaminidase A); and any variants thereof. The AAV vector also includes conventional control elements or sequences which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Suitable genes include those genes discussed in Anguela et al. “Entering the Modern Era of Gene Therapy”, Annual Rev. of Med. Vol. 70, pages 272-288 (2019) and Dunbar et al., “Gene comes of age ”, Science, Vol. 359, Issue 6372, eaan4672 (2018). [0068] Expression control sequences can be linked to the transgenes. Examples of expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart el al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the b-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 promoter. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied compounds, include, the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system [WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad. Sci. USA, 93 :3346-3351 (1996)], the tetracycline-repressible system [Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], the tetracycline- inducible system [Gossen et al., Science , 268: 1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)], the RU486-inducible system [Wang et al., Nat. Biotech. , 15:239- 243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)] and the rapamycin-inducible system [Magari et al., J. Clin. Invest., 100:2865-2872 (1997)]. Other types of inducible promoters, which may be useful in this context, are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. [0069] Optionally, the native promoter for the transgene may be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0070] The transgene may also include a gene operably linked to a tissue specific promoter. For instance, if expression in skeletal muscle is desired, a promoter active in muscle should be used. These include the promoters from genes e.g. encoding skeletal b-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with activities higher than naturally-occurring promoters (see Li et ah, Nat. Biotech. , 17:241-245 (1999)). Examples of promoters that are tissue-specific are known for liver (albumin, Miyatake et ah, J. Virol ., 71:5124- 32 (1997); hepatitis B virus core promoter, Sandig et ah, Gene Ther ., 3:1002-9 (1996); alpha- fetoprotein (AFP), Arbuthnot et ak, Hum. Gene Ther ., 7:1503-14 (1996)), bone osteocalcin (Stein et ak, Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein (Chen et ak, J. Bone Miner. Res., 11:654-64 (1996)), lymphocytes (CD2, Hansal et ak, J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptor chain), neuronal such as neuron-specific enolase (NSE) promoter (Andersen et ak, Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light- chain gene (Piccioli et ak, Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene (Piccioli et ak, Neuron, 15:373-84 (1995)), among others.

[0071] The recombinant AAV can be used to produce a protein of interest in vitro, for example, in a cell culture. For example, the AAV can be used in a method for producing a protein of interest in vitro, where the method includes providing a recombinant AAV comprising a nucleotide sequence encoding the heterologous protein; and contacting the recombinant AAV with a cell in a cell culture, whereby the recombinant AAV expresses the protein of interest in the cell. The size of the nucleotide sequence encoding the protein of interest can vary. For example, the nucleotide sequence can be at least about 0.1 kilobases (kb), at least about 0.2 kb, at least about 0.3 kb, at least about 0.4 kb, at least about 0.5 kb, at least about 0.6 kb, at least about 0.7 kb, at least about 0.8 kb, at least about 0.9 kb, at least about 1 kb, at least about 1.1 kb, at least about 1.2 kb, at least about 1.3 kb, at least about 1.4 kb, at least about 1.5 kb, at least about 1.6 kb, at least about 1.7 kb, at least about 1.8 kb, at least about 2.0 kb, at least about 2.2 kb, at least about 2.4 kb, at least about 2.6 kb, at least about 2.8 kb, at least about 3.0 kb, at least about 3.2 kb, at least about 3.4 kb, at least about 3.5 kb in length, at least about 4.0 kb in length, at least about 5.0 kb in length, at least about 6.0 kb in length, at least about 7.0 kb in length, at least about 8.0 kb in length, at least about 9.0 kb in length, or at least about 10.0 kb in length. In some embodiments, the nucleotide is at least about 1.4 kb in length.

[0072] The recombinant AAV can also be used to produce a protein of interest in vivo , for example in an animal, such as a mammal. Some embodiments provide a method for producing a protein of interest in vivo , where the method includes providing a recombinant AAV comprising a nucleotide sequence encoding the protein of interest; and administering the recombinant AAV to the subject, whereby the recombinant AAV expresses the protein of interest in the subject. The subject can be, in some embodiments, a non-human mammal, for example, a monkey, a dog, a cat, a mouse, or a cow. The size of the nucleotide sequence encoding the protein of interest can vary. For example, the nucleotide sequence can be at least about 0.1 kb, at least about 0.2 kb, at least about 0.3 kb, at least about 0.4 kb, at least about 0.5 kb, at least about 0.6 kb, at least about 0.7 kb, at least about 0.8 kb, at least about 0.9 kb, at least about 1 kb, at least about 1.1 kb, at least about 1.2 kb, at least about 1.3 kb, at least about 1.4 kb, at least about 1.5 kb, at least about 1.6 kb, at least about 1.7 kb, at least about 1.8 kb, at least about 2.0 kb, at least about 2.2 kb, at least about 2.4 kb, at least about 2.6 kb, at least about 2.8 kb, at least about 3.0 kb, at least about 3.2 kb, at least about 3.4 kb, at least about 3.5 kb in length, at least about 4.0 kb in length, at least about 5.0 kb in length, at least about 6.0 kb in length, at least about 7.0 kb in length, at least about 8.0 kb in length, at least about 9.0 kb in length, or at least about 10.0 kb in length. In some embodiments, the nucleotide is at least about 1.4 kb in length.

[0073] Of particular interest is the use of recombinant AAV to express one or more therapeutic proteins to treat various diseases or disorders. Non-limiting examples of the diseases include cancer such as carcinoma, sarcoma, leukemia, lymphoma; and autoimmune diseases such as multiple sclerosis. Non-limiting examples of carcinomas include esophageal carcinoma; hepatocellular carcinoma; basal cell carcinoma, squamous cell carcinoma (various tissues); bladder carcinoma, including transitional cell carcinoma; bronchogenic carcinoma; colon carcinoma; colorectal carcinoma; gastric carcinoma; lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung; adrenocortical carcinoma; thyroid carcinoma; pancreatic carcinoma; breast carcinoma; ovarian carcinoma; prostate carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; renal cell carcinoma; ductal carcinoma in situ or bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilm's tumor; cervical carcinoma; uterine carcinoma; testicular carcinoma; osteogenic carcinoma; epithelieal carcinoma; and nasopharyngeal carcinoma. Non-limiting examples of sarcomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelio sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Non-limiting examples of solid tumors include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Non-limiting examples of leukemias include chronic myeloproliferative syndromes; acute myelogenous leukemias; chronic lymphocytic leukemias, including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and acute lymphoblastic leukemias. Examples of lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitf s lymphoma; Hodgkin's lymphoma; and the like.

[0074] Other non-liming examples of the diseases that can be treated using the AAV vectors, recombinant viruses and methods disclosed herein include genetic disorders including sickle cell anemia, cystic fibrosis, lysosomal acid lipase (LAL) deficiency 1, Tay-Sachs disease, Phenylketonuria, Mucopolysaccharidoses, Glycogen storage diseases (GSD, e.g., GSD types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and XIV), Galactosemia, muscular dystrophy (e.g., Duchenne muscular dystrophy), and hemophilia such as hemophilia A (classic hemophilia) and hemophilia B (Christmas Disease), Wilson’s disease, Fabry Disease, Gaucher Disease hereditary angioedema (HAE), and alpha 1 antitrypsin deficiency. In addition, the AAV vectors, recombinant viruses and methods disclosed herein can be used to treat other disorders that can be treated by local expression of a transgene in the liver or by expression of a secreted protein from the liver or a hepatocyte.

[0075] The amount of the heterologous protein expressed in the subject (e.g., the serum of the subject) can vary. For example, in some embodiments the protein can be expressed in the serum of the subject in the amount of at least about 9 milligram (mg)/mL, at least about 10 mg/mL, at least about 11 mg/mL, at least about 12 mg/mL, at least about 13 mg/mL, at least about 14 mg/mL, at least about 15 mg/mL, at least about 16 mg/mL, at least about 17 mg/mL, at least about 18 mg/mL, at least about 19 mg/mL, at least about 20 mg/mL, at least about 21 mg/mL, at least about 22 mg/mL, at least about 23 mg/mL, at least about 24 mg/mL, at least about 25 mg/mL, at least about 26 mg/mL, at least about 27 mg/mL, at least about 28 mg/mL, at least about 29 mg/mL, at least about 30 mg/mL, at least about 31 mg/mL, at least about 32 mg/mL, at least about 33 mg/mL, at least about 34 mg/mL, at least about 35 mg/mL, at least about 36 mg/mL, at least about 37 mg/mL, at least about 38 mg/mL, at least about 39 mg/mL, at least about 40 mg/mL, at least about 41 mg/mL, at least about 42 mg/mL, at least about 43 mg/mL, at least about 44 mg/mL, at least about 45 mg/mL, at least about 46 mg/mL, at least about 47 mg/mL, at least about 48 mg/mL, at least about 49 mg/mL, or at least about 50 mg/mL. The protein of interest may be expressed in the serum of the subject in the amount of about 9 pg/mL, about 10 pg/mL, about 50 pg/mL, about 100 pg/mL, about 200 pg/mL, about 300 pg/mL, about 400 pg/mL, about 500 pg/mL, about 600 pg/mL, about 700 pg/mL, about 800 pg/mL, about 900 pg/mL, about 1000 pg/mL, about 1500 pg/mL, about 2000 pg/mL, about 2500 pg/mL, or a range between any two of these values. A skilled artisan will understand that the expression level in which a protein of interest is needed for therapeutic efficacy can vary depending on non-limiting factors, such as the particular protein of interest and the subject receiving the treatment, and an effective amount of the protein can be readily determined by a skilled artisan using conventional methods known in the art without undue experimentation.

[0076] Methods of Producing Gene Therapy Viruses or Adeno-Associated Virus

[0077] The present disclosure provides materials and methods for producing rGTV or rAAV virions in cells, such as insect cells, fungal or mammalian cells.

[0078] rAAV particles can also be produced using methods disclosed in various embodiments. In some instances, rAAV particles can be produced by using an insect or mammalian cell that stably expresses some of the necessary components for rAAV particle production. For example, a plasmid (or multiple plasmids) including AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of the cell. The insect, fungal, or mammalian cell can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the viral vector including the 5' and 3' AAV ITR (and the nucleotide sequence encoding the heterologous protein, if desired). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the rAAV. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce a host regulatory gene, rep gene, and cap gene into packaging cells. As yet another non-limiting example, both the viral vector containing the 5' and 3' AAV ITRs, the host regulatory gene, or the rep-cap genes can be stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the rAAV.

[0079] Methods of making AAV viral particles are described in e.g., U.S. Patent Nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191, US6566118, US8137948; or International Publication Nos. WO1996039530, W01998010088, WO1999014354, WO1999015685, WO1999047691, W02000055342, W02000075353, W02001023597, W02015191508, WO2019217513, W02018022608, WO2019222136, W02020232044,

WO20 19222132; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et ah, Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et ah, J. Vir.63:3822-8 (1989); Kajigaya et ah, Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et ah, J. Vir.66: 6922-30 (1992); Kimbauer et ah, Vir., 219:37-44 (1996); Zhao et ah, Vir.272:382-93 (2000); the contents of each of which are herein incorporated by reference in their entirety. Examples of rLV production are disclosed in US6797512, US9556438, US7250299, US8652837, US7083981, US11208669, EP1036182, WO1999031251, US8846385, US6428953, and WO2018204694; the contents of each of which are herein incorporated by reference in their entirety.

[0080] Cells such as, e.g., mammalian cell (e.g., human cell or non-human mammalian cell) are capable of generating rAAV. For example, cells are capable of generating rAAV when provided AAV helper functions, AAV non-helper functions, and a nucleotide sequence that the cells use to generate an AAV vector genome. In various embodiments, the AAV helper functions, AAV non-helper functions, and a nucleotide sequence that the cells use to generate rAAV are provided by a vector that is delivered to cell, for example, via transfection with transfection reagents, via transductions/infections with other recombinant viruses, by incorporating nucleotide sequences into the genomes of the cells, or by other methods. Examples of such cells include cell lines such as HEK293, HeLa, CHO, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells. [0081] The term “vector” is understood to refer to any genetic element, such as a plasmid, phage, transposon, cosmid, bacmid, mini-plasmid (e.g., plasmid devoid of bacterial elements), Doggybone DNA (e.g., minimal, closed-linear constructs), chromosome, virus, virion (e.g., baculovirus), etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.

[0082] The vector from which the cell generates an rAAV vector genome may contain a promoter and a restriction site downstream of the promoter to allow insertion of a polynucleotide encoding one or more proteins of interest, wherein the promoter and the restriction site are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. The vector may also contain a posttranscriptional regulatory element downstream of the restriction site and upstream of the 3' AAV ITR. The viral construct may further comprise a polynucleotide inserted at the restriction site and operably linked with the promoter, where the polynucleotide comprises the coding region of a protein of interest.

[0083] The term “AAV helper” refer to AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. Thus, AAV helper functions include both of the major AAV open reading frames (ORFs), rep and cap. The Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. The capsid (Cap) expression products supply necessary packaging functions. AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vector genomes.

[0084] In various embodiments, a vector providing AAV helper functions includes a nucleotide sequence(s) that encode capsid proteins or Rep proteins. The cap genes and/or rep gene from any AAV serotype (including, but not limited to, AAV1 (NCBI Reference Sequence No./Genbank Accession No. NC_002077.1), AAV2 (NCBI Reference Sequence No./Genbank Accession No. NC_001401.2), AAV3 (NCBI Reference Sequence No./Genbank Accession No. NC_001729.1), AAV3B (NCBI Reference Sequence No./Genbank Accession No. AF028705.1), AAV4 (NCBI Reference Sequence No./Genbank Accession No. NC_001829.1), AAV5 (NCBI Reference Sequence No./Genbank Accession No. NC_006152.1), AAV6 (NCBI Reference Sequence No./Genbank Accession No. AF028704-1), AAV7 (NCBI Reference Sequence No./Genbank Accession No. NC_006260.1), AAV8 (NCBI Reference Sequence No./Genbank Accession No. NC_006261.1), AAV9 (NCBI Reference Sequence No./Genbank Accession No. AX7S3250.1), AAV10 (NCBI Reference Sequence No./Genbank Accession No. AY631965.1), AAV11 (NCBI Reference Sequence No./Genbank Accession No. AY631966.1), AAV12 (NCBI Reference Sequence No./Genbank Accession No. DQ813647.1), AAV13 (NCBI Reference Sequence No./Genbank Accession No. EU28SS62.1), Bba21, Bba26, Bba27, Bba29, Bba30, Bba31, Bba32, Bba33, Bba34, Bba35, Bba36, Bba37, Bba38, Bba41, Bba42, Bba43, Bba44, Bcel4, Bcel5, Bcel6, Bcel7, Bcel8, Bce20, Bce35, Bce36, Bce39, Bce40, Bce41, Bce42, Bce43, Bce44, Bce45, Bce46, Bey20, Bey22, Bey23, Bma42, Bma43, Bpol, Bpo2, Bpo3, Bpo4, Bpo6, Bpo8, Bpol3, Bpol8, Bpo20, Bpo23, Bpo24, Bpo27, Bpo28, Bpo29, Bpo33, Bpo35, Bpo36, Bpo37, Brh26, Brh27, Brh28, Brh29, Brh30, Brh31, Brh32, Brh33, Bfml7, Bfml8, Bfm20, Bfm21, Bfm24, Bfm25, Bfm27, Bfm32, Bfm33, Bfm34, Bfm35, AAV-rhlO, AAV-rh39, AAV-rh43, AAVanc80L65, or any variants thereof) can be used herein to produce the recombinant AAV Exemplary capsids are also provided in International Application No. WO 2018/022608 and WO 2019/222136, which are incorporated herein in its entirety. Each NCBI Reference Sequence Number or Genbank Accession Numbers provided above is also incorporated by reference herein. In some embodiments, the AAV cap genes encode a capsid from serotype 1, serotype 2, serotype 3, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13, or a variant thereof.

[0085] For production, cells with AAV helper functions produce recombinant capsid proteins sufficient to form a capsid. This includes at least VPl and VP3 proteins, but more typically, all three of VPl, VP2, and VP3 proteins, as found in native AAV. The sequence of the capsid proteins determines the serotype of the AAV virions produced by the host cell. Capsids useful in the invention include those derived from a number of AAV serotypes, including 1, 2, 3, 3B, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13 or mixed serotypes (see, e.g., US PatentNo. 8318480 for its disclosure of non natural mixed serotypes). The capsid proteins can also be variants of natural VPl, VP2 and VP3, including mutated, chimeric or shuffled proteins. The capsid proteins can be those of rh.lO or other subtype within the various clades of AAV; various clades and subtypes are disclosed, for example, in U.S. Patent No. 7,906,111. Because of wide construct availability and extensive characterization, illustrative AAV vectors disclosed below are derived from serotype 2. Construction and use of AAV vectors and AAV proteins of different serotypes are discussed in Chao et ah, Mol. Ther. 2:619-623, 2000; Davidson et ah, PNAS 97:3428-3432, 2000; Xiao et ah, J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol. 74:1524-1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, 2001. In various embodiments, nucleotide sequences encoding VP proteins can be operably linked to a suitable expression control sequence. For example, the nucleotide sequences can be operably linked to eukaryotic promoters.

[0086] For production, cells with AAV helper functions produce Rep proteins to promote production of rAAV. It has been found that infectious particles can be produced when at least one large Rep protein (Rep78 or Rep68) and at least one small Rep protein (Rep52 and Rep40) are expressed in cells. In a specific embodiment all four of Rep 78, Rep68, Rep52 and Rep 40 are expressed. Alternately, Rep78 and Rep52, Rep78 and Rep40, Rep 68 and Rep52, or Rep68 and Rep40 are expressed. Examples below demonstrate the use of the Rep78/Rep52 combination. Rep proteins can be derived from AAV-2 or other serotypes. In various embodiments, nucleotide sequences encoding Rep proteins can be operably linked to a suitable expression control sequence such as eukaryotic promoters. For example, the nucleotide sequences can be operably linked to eukaryotic promoters.

[0087] For production, cells with AAV helper functions produce Rep proteins to promote production of rAAV. It has been found that infectious particles can be produced when at least one large Rep protein (Rep78 or Rep68) and at least one small Rep protein (Rep52 and Rep40) are expressed in cells. In a specific embodiment all four of Rep 78, Rep68, Rep52 and Rep 40 are expressed. Alternately, Rep78 and Rep52, Rep78 and Rep40, Rep 68 and Rep52, or Rep68 and Rep40 are expressed. Examples below demonstrate the use of the Rep78/Rep52 combination. Rep proteins can be derived from AAV-2 or other serotypes. In various embodiments, nucleotide sequences encoding Rep proteins can be operably linked to a suitable expression control sequence such as eukaryotic promoters. For example, the nucleotide sequences can be operably linked to eukaryotic promoters.

[0088] In order to promote viral assembly, the host cell expresses recombinant assembly activating protein (AAP). The AAP expressing gene can be linked to suitable expression control sequences and encoded on plasmids or integrated into a yeast cell chromosome. AAP can be derived from AAV-2 or other serotypes. [0089] In addition to the capsid, Rep, and AAP genes, embodiments include exogenous polynucleotides that express helper proteins. Without limitation, helper gene products that can be expressed in the host cell in various combinations include Spodoptera frugiperda FKBP46, human FKBP52, Adenovirus El A, E1B, E2A, E4 and VA, Herpes simplex virus UL29, UL30, UL42, U15, UL8, UL52, and UL9. In an embodiment, the cell expresses at least one immunophilin analogue (i.e., an immunophilin or similar protein) and at least one helper virus gene product.

[0090] The term “non-AAV helper function” refers to non-AAV derived viral and/or cellular functions upon which AAV is dependent for its replication. Thus, the term captures proteins and RNAs that are required in AAV replication, including those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) and vaccinia virus.

[0091] The term “non-AAV helper function vector” refers generally to a nucleic acid molecule that includes nucleotide sequences providing accessory functions. An accessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting AAV virion production in the host cell. Expressly excluded from the term are infectious viral particles as they exist in nature, such as adenovirus, herpesvirus or vaccinia virus particles. Thus, accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid. In particular, it has been demonstrated that the full-complement of adenovirus genes are not required for accessory helper functions. For example, adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al, (1971) Virology 45:317. Similarly, mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions. Carter et al., (1983) Virology 126:505. However, adenoviruses defective in the El region, or having a deleted E4 region, are unable to support AAV replication. Thus, El A and E4 regions are likely required for AAV replication, either directly or indirectly. Laughlin et al., (1982). J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983) Virology 126:505. Other characterized Ad mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239;

Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981)

Proc. Natl. Acad. Sci. USA 78:2927; Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)). Although studies of the accessory functions provided by adenoviruses having mutations in the E1B coding region have produced conflicting results, Samulski et al., (1988) J. Virol. 62:206- 210, recently reported that ElB55k is required for AAV virion production, while ElB19k is not. In addition, International Publication WO 97/17458 and Matshushita et al., (1998) Gene Therapy 5:938-945, describe accessory function vectors encoding various Ad genes. Particularly preferred accessory function vectors comprise an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2A 72 kD coding region, an adenovirus El A coding region, and an adenovirus E1B region lacking an intact ElB55k coding region. Such vectors are described in International Publication No. WO 01/83797.

[0092] The host cells can be transfected using various transfection reagents such as cationic peptides.

[0093] The host cells can be transformed to achieve stably maintained episomes or chromosomal integration of various recombinant genetic elements. Homologous recombination from a vector such as a plasmid can be used for chromosomal integration. See, e.g., Rothstein, R., “Targeting, disruption, replacement, and allele rescue: Integrative DNA transformation in yeast” Methods in Enzymology vol. 194, 1991, pp. 281-301. For example, a VP, Rep, AAP or helper transgene can be fused to a selection marker such as a hygromycin resistance gene (hph) with a flanking sequence targeting a neutral site such as TY retrotransposon (i.e., a site that when disrupted does not unduly interfere with the vitality of the host cell). This can be repeated for different genes with additional selection markers and integration sites.

[0094] In various embodiments, methods of generating a gene therapy vector comprise the step of culturing a host cell with the gene therapy vector production culture medium of various embodiments wherein the host cells comprise one or more vectors for gene therapy vector production. [0095] The phrase “gene therapy vector” refers to viral vectors. The viral vector may be an adeno-associated virus (AAV), an adenovirus, a retrovirus, a pox virus, a lentivirus, , a vaccinia virus, or a herpes simplex virus. The gene therapy vector expresses a biologic.

[0096] In various embodiments, methods of generating rAAV comprise the step of culturing a host cell with the cell culture medium of various embodiments. The host cells comprise one or more vectors for rAAV production.

[0097] In various embodiments, methods of generating rAAV comprise the steps of culturing a host cell comprising one or more vectors for rAAV production in a cell culture medium and adding the feed formulation of various embodiments to the cell culture medium at a predetermined rate such that the cell culture media comprises a nicotinamide concentration of 1 mM or more for a predetermined time.

[0098] In various embodiments, the host cell is transfected with the one or more vectors for rAAV production prior to the culturing step.

[0099] In various embodiments, the feed formulation is added before or after the transfecting step, preferably 2 or more hours after the transfecting step.

[00100] For rAAV feed development, a combination of multiple components supplying different metabolic pathways will have a positive effect on the titers, especially when compared to a single component. Theoretical knowledge in the art indicates that viral vector production is a complex process requiring synthesis of DNA and viral proteins using the cellular machinery as well as nutrient consumption for normal cell growth and proliferation. Such approach was applied for the feed development of enveloped viruses (Rodrigues AF, Formas-Oliveira AS, Bandeira VS, Alves PM, Hu WS, Coroadinha AS, “Metabolic pathways recruited in the production of a recombinant enveloped virus: mining targets for process and cell engineering.” Metabolic engineering 2013 , 20:131-145).

[00101] Individual components were selected based on accumulating knowledge on the metabolic pathways involved in the cell growth and proliferation and rAAV production. Below the role of individual components are discussed in more detail.

[00102] The amino acid profile of the production media of choice for HEK293 cells culture was measured during rAAV production in a 3L bioreactor (Eppendorf BioBlu 3c). Serine and arginine were found to be completely depleted sometime after transfection and therefore included into the feed. Serine consumption with the corresponding glycine production is an indication of an active folic acid cycle (Koseki J, Konno M, Asai A, Colvin H, Kawamoto K, Nishida N, Sakai D, Kudo T, Satoh T, Doki Y et ah, “Enzymes of the one-carbon folate metabolism as anticancer targets predicted by survival rate analysis.” Scientific reports 2018, 8(1):303), which is a pathway for rapid proliferation. In the exemplary formulations of Tables 1 and 2, the final concentrations of serine and arginine are each 5 mM.

[00103] Folic acid is a vitamin that is poorly soluble in water and degrades with exposure to light and high temperature (Gazzali AM, Lobry M, Colombeau L, Acherar S, Azais H, Mordon S, Arnoux P, Baros F, Vanderesse R, Frochot C, “Stability of folic acid under several parameters.” European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 2016, 93:419-430). Therefore, folic acid is added in the feed in the form of a vitamins supplement (e.g., Gibco, cat# 11120-052). In the exemplary formulations of Tables 1 and 2, the vitamins supplement is included in an amount of 10 mL/L from 100.0 mg/L Choline chloride; 100.0 mg/L D-Calcium pantothenate; 100 mg/L Folic Acid; 100 mg/L Nicotinamide; 100 mg/L Pyridoxal hydrochloride; 100 mg/L Riboflavin; 100 mg/L Thiamine hydrochloride; and 200 mg/L I-Inositol.

[00104] A study was performed to understand the effect of several cell culture additives on the HEK293 cells metabolism. Ornithine was identified as a component significantly affecting the glutamine levels in the spent medum. It was hypothesized that glutamine was partially consumed for polyamine biosynthesis as it appeared to be sensitive to ornithine supplementation. Consumption of arginine also might be linked partially to the polyamine biosynthesis (Albaugh VL, Pinzon-Guzman C, Barbul A, “Arginine-Dual roles as an onconutrient and immunonutrient ” Journal of surgical oncology 2017, 115(3):273-280). Therefore, ornithine in the form of LOLA is included into the feed formulation of the present invention. In the exemplary formulations of Tables 1 and 2, the final concentration of LOLA is 3 mM.

[00105] REP protein, which is essential for DNA replication and packaging of adeno- associated viral DNA, has several known enzymatic functions: helicase, nickase, and DNA - binding. Helicase activity requires hydrolysis of ATP to ADP. Overexpression of ATPase would require consumption of ATP -generating substrates to keep the energy charge of the cell constant. Historically, glucose and glutamine are added to the cell culture media as main energy substrates. However, their rapid and inefficient consumption by cancer cells may limit energy dependent processes like protein, lipid, and DNA synthesis. To overcome this potential limitation, several ATP-generating substrates were tested as supplements: pyruvate plus Diehl oroacetate (DCA), fatty acids plus carnitine, branched chain keto acids (BCKA), and Beta hydroxy butyrate (BHB). Although any of the above substrates can be used, experiments suggested that fatty acid plus carnitine had a positive effect on the rAAV titers. Therefore, this substrate was chosen to be included in the exemplary feed of the present invention. In the exemplary formulations of Table

1, the final concentration of carnitine is 10 mM. In the exemplary formulations of Table 1, the fatty acid supplement is included in an amount of 0.5 mL/L.

[00106] Overexpression of REP78 protein in the HEK293 cells was found to deplete cellular levels of NAD/NADH, a reducing agent involved in numerous redox reactions. It was hypothesized that nickase activity of the REP is responsible for activation of PARP (Poly ADP Ribose Polymerase), an enzyme which consumes NAD as a substrate in an attempt to repair cellular DNA (Murata MM, Kong X, Moncada E, Chen Y, Wang P, Berns MW, Yokomori K, Digman MA, “NAD consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival.” 2018). Overexpression of REP78 coincides with rapid cell death. To mitigate the reduction of cellular NAD during rAAV production, two approaches were tested: supplementation of the substrate (nicotinamide mono-nucleotide, MNM) for NAD synthesis and providing endogenous PARP inhibitors (nicotinamide (NAM), taurine) (Banasik M, Stedeford T, Strosznajder RP, “Natural inhibitors of poly(ADP-ribose) polymerase-1.” Molecular neurobiology 2012, 46(l):55-63). Although either approach may be used, the addition of nicotinamide and taurine is preferred. In the exemplary formulations of Tables 1 and

2, the final concentration of taurine is 30 mM, and the final concentration of NAM is 10 mM or 20 mM.

[00107] Supplementation of the nicotinamide, a precursor of the NAD biosynthesis (Audrito V, Manago A, Gaudino F, Sorci L, Messana VG, Raffaelli N, Deaglio S, “NAD-Biosynthetic and Consuming Enzymes as Central Players of Metabolic Regulation of Innate and Adaptive Immune Responses in Cancer.” Frontiers in Immunology 2019, 10) and PARP inhibitor (Banasik M, Stedeford T, Strosznajder RP, “Natural inhibitors of poly(ADP-ribose) polymerase-1.” Molecular neurobiology 2012, 46(l):55-63), helps to mitigate the cytotoxic effect of the AAV REP protein expression and increase cellular productivity. Activation of PARP 1 DNA repair pathways and depletion of cellular NAD pool was reported in case of other viruses such as the herpes virus (Grady SL, Hwang J, Vastag L, Rabinowitz JD, Shenk T, “Herpes Simplex Virus 1 Infection Activates Poly(ADP-Ribose) Polymerase and Triggers the Degradation of Poly(ADP- Ribose) Glycohydrolase.” Journal of Virology 2012, 86(15):8259-8268) and inhibition of PARP was reported to increase adenoviral titers (Nebenzahl-Sharon K, Sharf R, Amer J, Shalata H, Khoury-Haddad H, Sohn S-Y, Ayoub N, Hearing P, Kleinberger T, Banks L, “An Interaction with PARP-1 and Inhibition of Parylation Contribute to Attenuation of DNA Damage Signaling by the Adenovirus E4orf4 Protein.” Journal of Virology 2019, 93(19)). However, the concept of NAD depletion or PARP activation upon rAAV infection/production is not known in the art.

[00108] Cholesterol is added to the feed formulation of the present invention based on the assumption that cholesterol will enhance the endocytosis of the transfection complexes (Zuhorn IS, Kalicharan R, Hoekstra D, “Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis.” The Journal of biological chemistry 2002, 277(20):18021-18028). In general cholesterol can be supplied as free cholesterol, cholesterol complexed with cyclodextrin or any other water soluble form. The cholesterol may be added in the form of a cholesterol supplement. In the exemplary formulations of Table 1, the cholesterol supplement is included in an amount of 2 ml/L. Other cholesterol concentration are disclosed in W02013006461 and Zuhorn et ah, Journal of Biological Chemistry 277.20 (2002): 18021-18028, both of which are incorporated by reference in their entirety.

[00109] To increase AAV production, the feed formulation of the present invention is added to host cells in a cell culture media. In general, infectious rAAV particles are produced in a “host cell.” The practice of the present methods will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, cell culture, virology, and the like which are in the skill of one in the art. These techniques are fully disclosed in current literature and reference is made specifically to Sambrook, Fritsch and Maniatis eds., “Molecular Cloning, A Laboratory Manual”, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Celis J. E. “Cell Biology, A Laboratory Handbook” Academic Press, Inc. (1994) and Bahnson et ah, J. of Virol. Methods, 54:131-143 (1995). Furthermore, all publications and patent applications cited in this specification are indicative of the level of skill of those skilled in the art to which these methods pertain and are hereby incorporated by reference in their entirety.

[00110] The time when the feed formulation of the present invention is added is not particularly limited, and the feed formulation of the present invention can be added at any time. However, the feed formulation of the present invention is preferably added after transfection. In particular, the feed formulation of the present invention is added 2-48 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, or 48 hours) post transfection.

[00111] The cell culture media may be chosen based on the type of host cell as would be understood by one of ordinary skill in the art.

[00112] In various embodiments, the culturing step of any aspect or embodiment occurs in a volume of at least 5 milliliter(s) (mL), at least 10 mL, at least 20 mL, at least 50 mL, at least 100 mL, at least 500 mL, at least 1 liter (L), at least 10 L, at least 50 L, at least 100L, at least 250 L, at least 500 L, at least 1000 L, at least 1500 L, at least 2000 L, or at least 2500 L.

[00113] In examples, the culturing step can occur in a spin tube or shake flask(s). In various embodiments, the culturing step of any aspect or embodiment occurs in a volume of 5 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L, or 5 L. In other embodiments, the volume of the culturing step is a range between any two volumes provided above.

[00114] In other examples, the culturing step can occur in a bioreactor or bioreactors. In various embodiments, the culturing step of any aspect or embodiment occurs in a volume of 1 L, 2 L, 3 L, 4 L, 5 L, 6L, 7L, 8 L, 9 L, 10 L, 11 L, 12 L, 13 L, 14 L, 15 L, 16 L, 17 L, 18 L, 19 L, 20 L, 21 L, 22 L, 23 L, 24 L, 25 L, 26 L, 27 L, 28 L, 29 L, 30 L, 31 L, 32 L, 33 L, 34 L, 35 L, 36

L, 37 L, 38 L, 39 L, 40 L, 41 L, 42 L, 43 L, 44 L, 45 L, 46 L, 47 L, 48 L, 49 L, 50 L, 51 L, 52 L,

53 L, 54 L, 55 L, 56 L, 57 L, 58 L, 59 L, 60 L, 61 L, 62 L, 63 L, 64 L, 65 L, 66 L, 67 L, 68 L, 69

L, 70 L, 71 L, 72 L, 73 L, 74 L, 75 L, 76 L, 77 L, 78 L, 79 L, 80 L, 81 L, 82 L, 83 L, 84 L, 85 L, 86 L, 87 L, 88 L, 89 L, 90 L, 91 L, 92 L, 93 L, 94 L, 95 L, 96 L, 97 L, 98 L, 99 L, 100 L, 110 L, 120 L, 130 L, 140 L, 150 L, 160 L, 170 L, 180 L, 190 L, 200 L, 210 L, 220 L, 230 L, 240 L, 250 L, 260 L, 270 L, 280 L, 290 L, 300 L, 310 L, 320 L, 330 L, 340 L, 350 L, 360 L, 370 L, 380 L, 390 L, 400 L, 410 L, 420 L, 430 L, 440 L, 450 L, 460 L, 470 L, 480 L, 490 L, 500 L, 510 L, 520 L, 530 L, 540 L, 550 L, 560 L, 570 L, 580 L, 590 L, 600 L, 610 L, 620 L, 630 L, 640 L, 650 L, 660 L, 670 L, 680 L, 690 L, 700 L, 710 L, 720 L, 730 L, 740 L, 750 L, 760 L, 770 L, 780 L, 790 L, 800 L, 810 L, 820 L, 830 L, 840 L, 850 L, 860 L, 870 L, 880 L, 890 L, 900 L, 910 L, 920 L, 930 L, 940 L, 950 L, 960 L, 970 L, 980 L, 990 L, 1000 L, 1010 L, 1020 L, 1030 L, 1040 L, 1050 L, 1060 L, 1070 L, 1080 L, 1090 L, 1100 L, 1110 L, 1120 L, 1130 L, 1140 L, 1150 L, 1160 L, 1170 L, 1180 L, 1190 L, 1200 L, 1210 L, 1220 L, 1230 L, 1240 L, 1250 L, 1260 L, 1270 L, 1280 L, 1290 L, 1300 L, 1310 L, 1320 L, 1330 L, 1340 L, 1350 L, 1360 L, 1370 L, 1380 L, 1390 L, 1400 L, 1410 L, 1420 L, 1430 L, 1440 L, 1450 L, 1460 L, 1470 L, 1480 L, 1490 L, 1500 L, 1510 L, 1520 L, 1530 L, 1540 L, 1550 L, 1560 L, 1570 L, 1580 L, 1590 L, 1600 L, 1610 L, 1620 L, 1630 L, 1640 L, 1650 L, 1660 L, 1670 L, 1680 L, 1690 L, 1700 L, 1710 L, 1720 L, 1730 L, 1740 L, 1750 L, 1760 L, 1770 L, 1780 L, 1790 L, 1800 L, 1810 L, 1820 L, 1830 L, 1840 L, 1850 L, 1860 L, 1870 L, 1880 L, 1890 L, 1900 L, 1910 L, 1920 L, 1930 L, 1940 L, 1950 L, 1960 L, 1970 L, 1980 L, 1990 L, 2000 L, 2010 L, 2020 L, 2030 L, 2040 L, 2050 L, 2060 L, 2070 L, 2080 L, 2090 L, 2100 L, 2110 L, 2120 L, 2130 L, 2140 L, 2150 L, 2160 L, 2170 L, 2180 L, 2190 L, 2200 L, 2210 L, 2220 L, 2230 L, 2240 L, 2250 L, 2260 L, 2270 L, 2280 L, 2290 L, 2300 L, 2310 L, 2320 L, 2330 L, 2340 L, 2350 L, 2360 L, 2370 L, 2380 L, 2390 L, 2400 L, 2410 L, 2420 L, 2430 L, 2440 L, 2450 L, 2460 L, 2470 L, 2480 L, 2490 L, 2500 L, 2510 L, 2520 L, 2530 L, 2540 L, 2550 L, 2560 L, 2570 L, 2580 L, 2590 L, 2600 L, 2610 L, 2620 L, 2630 L, 2640 L, 2650 L, 2660 L, 2670 L, 2680 L, 2690 L, 2700 L, 2710 L, 2720 L, 2730 L, 2740 L, 2750 L, 2760 L, 2770 L, 2780 L, 2790 L, 2800 L, 2810 L, 2820 L, 2830 L, 2840 L, 2850 L, 2860 L, 2870 L, 2880 L, 2890 L, 2900 L, 2910 L, 2920 L, 2930 L, 2940 L, 2950 L, 2960 L, 2970 L, 2980 L, 2990 L, or 3000 L. In other embodiments, the volume of the culturing step is a range between any two volumes provided above.

[00115] rAAV particles can also be produced using methods disclosed in various embodiments. In some instances, rAAV particles can be produced by using an insect, fungalor mammalian cell that stably expresses some of the necessary components for rAAV particle production. For example, a plasmid (or multiple plasmids) including AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of the cell. The insect, fungal, or mammalian cell can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the viral vector including the 5' and 3' AAV ITR (and the nucleotide sequence encoding the heterologous protein, if desired). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the rAAV. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce a host regulatory gene, rep gene, and cap gene into packaging cells. As yet another non-limiting example, both the viral vector containing the 5' and 3' AAV ITRs, the host regulatory gene, or the rep-cap genes can be stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the rAAV.

[00116] Other aspects and advantages of the present disclosure will be understood upon consideration of the following illustrative examples.

EXAMPLES Example 1

[00117] A combination of the compounds listed in Table 1 was tested against commercially available feeds for recombinant protein production. The feed formulation of Table 1 demonstrated comparable or better titers.

[00118] The effect of the feed formulation in comparison with several other commercially available feeds was assessed in terms of rAAV titers produced in shake flasks. The rAAV capsids that were tested were the Bba41 capsids. The feed formulation of Table 1 was compared to other supplements. HEK293 cells were transfected with plasmids for rAAV production using a cationic peptide transfection reagent, and feeds were added post transfection to all conditions. The feed formulation of various embodiments and the cell culture medium of various embodiments showed increased or comparable production of Bba41 capsids as compared to other commercially available feeds.

Example 2

[00119] The feed formulation of Table 1 was successfully tested at high cell densities at transfection where nutrients limitation plays a greater role in rAAV production. In this regard, Figure 1 shows the effect of the feed formulation added post transfection of Table 1 on Bba41 titers at high cell density. HEK293 cells were cultured in media of choice and transfected with plasmids for rAAV production using a cationic peptide transfection reagent. Figure 1 shows the improvement in titer of Bba41 capsids using the feed formulation of various embodiments and the cell culture medium of various embodiments.

Example 3

[00120] The feed formulation of Table 1 was then tested with another capsid (AAV9) and another production media of choice demonstrating higher rAAV titers in comparison to no feed conditions. HEK293 cells were transfected with plasmids for rAAV production using a cationic transfection reagent. The feed formulation of various embodiments and the cell culture medium of various embodiments showed increased production of AAV9 capsids as compared to no feeds condition.

Example 4

[00121] The feed formulation of Table 2 was tested with AAV9 capsids in HEK293 cellsin Ambrl5 mini bioreactors with different cell densities at transfection. HEK293 cells were transfected with plasmids for rAAV production using a cationic transfection reagent. The feed formulation of Table 2 was added post transfection. When compared to the “no feed” condition, the feed formula of Table 2 with 20 mM NAM showed increased AAV9 titers titer at different cell densities. The results are shown in FIG. 2. Thus, the feed formulation of various embodiments and the cell culture medium of various embodiments showed increased production of AAV9 capsids.

[00122] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.