Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells

<div>Increased VL30 genome copy number (GCN) in 293T cells, which were cocultured with PG13 cells. (a) Physical map of pTK1261 expressing the firefly luciferase and the GFP-blasticidin selection marker under transcriptional and translational control of a CMV promoter and an internal ribosome entry site (IRES), respectively. (b) Bar graph showing VL30 GCN in single-cell clones of 293T cells. PG13 and pTK2161 vector-transduced 293T cells were cocultured for 7 passages. Single-cell clones of cocultured 293T cells were isolated in the presence of blasticidin (<span class="nowrap"><svg height="12.7373pt" id="M66" style="vertical-align:-3.429399pt" version="1.1" viewbox="-0.0498162 -9.3079 41.3167 12.7373" width="41.3167pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.013,0,0,-0.013,0,0)"><path d="M153 550H386L412 615L406 623H120L82 318C104 327 142 338 184 338C294 338 347 275 347 187C347 112 305 39 221 39C160 39 119 71 97 89C88 97 80 96 71 90C59 80 50 67 49 57C48 45 52 36 66 23C80 9 123 -12 169 -12C221 -11 288 15 342 59C403 109 431 165 431 225C431 308 366 395 238 395C212 395 165 379 127 364L153 550Z"></path></g><g transform="matrix(.013,0,0,-0.013,8.416,0)"><path d="M538 96L524 119C492 88 454 63 446 63C439 63 434 70 440 101C463 223 491 341 518 448H508L433 422L401 276C355 192 240 56 188 56C163 56 154 89 163 133C184 233 207 338 235 443L230 448L152 424L58 17C40 -60 23 -143 23 -185C23 -241 42 -261 63 -261C92 -261 117 -241 125 -227L124 -221C109 -209 89 -165 89 -103C89 -55 96 -13 105 26H107C121 -3 134 -12 151 -12C172 -12 194 -5 221 16C279 61 330 124 384 194H386C381 159 377 141 368 105C343 3 363 -12 383 -12C416 -12 486 35 538 96Z"></path></g><g transform="matrix(.013,0,0,-0.013,15.632,0)"><path d="M463 437C426 431 375 425 327 422C297 440 264 449 231 449H230C153 449 51 396 51 283C51 215 94 168 139 149C123 129 91 103 51 88C50 78 53 60 62 46C75 25 100 2 136 -9C112 -28 73 -59 53 -79C38 -94 29 -113 29 -135C30 -192 91 -257 203 -257C336 -257 452 -160 452 -59C452 39 371 59 309 59C275 59 240 58 203 58C158 58 140 77 140 96C140 110 157 129 170 138C186 135 205 133 221 133C306 133 396 185 396 293C396 328 384 360 366 381L423 378C439 387 459 413 468 429L463 437ZM219 418C277 418 314 362 314 284C314 205 275 166 231 165C176 165 137 221 137 299C137 376 177 418 219 418ZM241 -11C285 -11 314 -14 339 -24C367 -36 384 -61 384 -95C384 -157 335 -206 240 -206C166 -206 108 -165 108 -110C108 -82 128 -54 154 -32C172 -17 195 -11 241 -11Z"></path></g><g transform="matrix(.013,0,0,-0.013,21.738,0)"><path d="M368 703H309L44 -163H104L368 703Z"></path></g><g transform="matrix(.013,0,0,-0.013,27.1,0)"><path d="M797 0V26C739 32 732 36 732 103V296C732 394 682 449 605 449C576 449 550 437 529 423C504 407 475 389 446 366C425 418 382 449 334 449C303 449 279 437 253 421C222 403 201 385 180 371V452C135 432 85 419 41 411V388C99 379 102 374 102 310V103C102 38 93 32 27 26V0H238V26C189 32 180 38 180 103V338C210 363 250 390 289 390C351 390 377 348 377 275V103C377 37 368 32 306 26V0H520V26C465 32 456 38 456 101V296C456 314 455 326 453 338C491 369 529 390 565 390C628 390 653 345 653 274V107C653 36 642 32 583 26V0H797Z"></path></g><g transform="matrix(.013,0,0,-0.013,37.812,0)"><path d="M238 0V26C174 32 166 38 166 104V712C132 700 70 683 18 677V653C81 647 87 645 87 577V104C87 38 78 32 15 26V0H238Z"></path></g></svg>).</span> GCN in each cell clone was determined by qPCR. pTK2161-transduced 293T served as negative control. The experiment was performed in technical triplicate.</div>

Advances in Cell and Gene Therapy

Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells

Figure 3