3 Tiers for AAV Po­ten­cy As­say De­vel­op­ment Suc­cess

Ade­no-as­so­ci­at­ed virus (AAV) gene ther­a­pies rep­re­sent a ma­jor ad­vance­ment in treat­ing ge­net­ic dis­or­ders by de­liv­er­ing ge­net­ic ma­te­r­i­al di­rect­ly to pa­tients’ cells. These ther­a­pies of­fer the po­ten­tial for cu­ra­tive treat­ments by tar­get­ing spe­cif­ic ge­net­ic de­fects. How­ev­er, their com­plex­i­ty ne­ces­si­tates rig­or­ous test­ing to en­sure ef­fi­ca­cy. Among the var­i­ous types of as­says used, po­ten­cy as­says stand out as crit­i­cal for un­der­stand­ing both the qual­i­ty and strength of AAV ther­a­pies. These as­says not on­ly mea­sure bi­o­log­i­cal ac­tiv­i­ty but al­so serve as in­di­ca­tors of sta­bil­i­ty, of­fer­ing in­sight in­to the prod­uct’s long-term ef­fec­tive­ness. Due to their sig­nif­i­cance, po­ten­cy as­says are of­ten a pri­ma­ry fo­cus for reg­u­la­to­ry agen­cies like the FDA and EMA, which re­quire well-de­vel­oped po­ten­cy as­says to ap­prove AAV prod­ucts. Forge Bi­o­log­ics sup­ports de­vel­op­ers across all clin­i­cal phas­es by of­fer­ing a tiered ap­proach to po­ten­cy as­say de­vel­op­ment, qual­i­fi­ca­tion, and val­i­da­tion, fa­cil­i­tat­ing the pro­gres­sion of safe and ef­fec­tive ther­a­pies to mar­ket.

Reg­u­la­to­ry Guid­ance on Po­ten­cy As­say De­sign and De­vel­op­ment is Evolv­ing Re­quir­ing Adapt­able Strate­gies

As AAV gene ther­a­pies ad­vance through clin­i­cal tri­als and reach the mar­ket, reg­u­la­to­ry guid­ance on po­ten­cy as­say re­quire­ments con­tin­ues to evolve. The FDA’s 2011 Guid­ance for In­dus­try and the In­ter­na­tion­al Coun­cil for Har­mon­i­sa­tion’s (ICH) Q6B guide­lines pro­vide com­pre­hen­sive frame­works for po­ten­cy as­says, with re­cent up­dates em­pha­siz­ing as­say qual­i­fi­ca­tion and val­i­da­tion through­out the prod­uct life­cy­cle¹. While ear­ly-phase clin­i­cal tri­als may al­low more flex­i­bil­i­ty, re­quire­ments be­come in­creas­ing­ly strin­gent as prod­ucts ad­vance to­wards com­mer­cial­iza­tion.

In late 2023, the FDA re­leased a draft guid­ance ti­tled, “Po­ten­cy As­sur­ance for Cel­lu­lar and Gene Ther­a­py Prod­ucts,” which fur­ther clar­i­fies ex­pec­ta­tions for po­ten­cy as­says². This guid­ance high­lights the need for po­ten­cy as­says to mea­sure bi­o­log­i­cal ac­tiv­i­ty and en­sure prod­uct con­sis­ten­cy and sta­bil­i­ty, el­e­vat­ing their role in the over­all qual­i­ty con­trol strat­e­gy for gene ther­a­py prod­ucts.

Com­plex­i­ties of AAV Po­ten­cy As­say De­vel­op­ment

De­vel­op­ing po­ten­cy as­says for AAV ther­a­pies re­quires thought­ful ap­proach­es to nav­i­gate com­mon chal­lenges. Ear­ly-phase as­says fo­cus on proof-of-con­cept, of­ten us­ing in vit­ro re­porter as­says in­volv­ing trans­fec­tion with plas­mids en­cod­ing a gene of in­ter­est (GOI). These as­says are quick and flex­i­ble, al­low­ing ad­just­ments as un­der­stand­ing of the prod­uct’s mech­a­nism of ac­tion (MOA) evolves.

As pro­grams move in­to Phase I/II clin­i­cal tri­als, the fo­cus shifts to de­vel­op­ing quan­ti­ta­tive as­says that re­li­ably mea­sure bi­o­log­i­cal ac­tiv­i­ty and be­gin to de­fine po­ten­cy cri­te­ria. This process re­quires time and cre­ativ­i­ty due to vari­abil­i­ty in start­ing ma­te­ri­als like nov­el vec­tors, trans­genes, and cell types, with vec­tor de­sign–serotype, pro­mot­er, GOI, etc.—all fo­cused on en­sur­ing ef­fi­cient, tar­get­ed, and safe in vi­vo de­liv­ery.  How­ev­er, AAV trans­duc­tion mech­a­nisms re­main un­clear, and in vit­ro cul­tures lack the cel­lu­lar di­ver­si­ty and ex­tra­cel­lu­lar sup­port found in vi­vo, lead­ing to dif­fer­ences in tro­pism and trans­duc­tion ef­fi­cien­cy³.

A func­tion­al in vit­ro po­ten­cy as­say de­pends on se­lect­ing a cell line that ex­press­es vec­tor-de­rived RNA un­der the cho­sen pro­mot­er, and, if ap­plic­a­ble, trans­lates it in­to a func­tion­al pro­tein. Once a suit­able cell line is iden­ti­fied, trans­duc­tion con­di­tions are op­ti­mized for the vec­tor serotype. Since AAV ther­a­pies of­ten tar­get in­tri­cate bi­o­log­i­cal path­ways, un­der­stand­ing or defin­ing the MOA may be dif­fi­cult. De­vel­op­ing, qual­i­fy­ing, and val­i­dat­ing func­tion­al po­ten­cy as­says that re­flect the MOA is es­sen­tial as pro­grams progress to­wards com­mer­cial­iza­tion.

Forge Bi­o­log­ics’ Tiered Ap­proach to Po­ten­cy As­say De­vel­op­ment and Qual­i­fi­ca­tion

Forge Bi­o­log­ics of­fers a tiered ap­proach to de­vel­op­ing quan­ti­ta­tive po­ten­cy as­says, pro­vid­ing flex­i­ble sup­port to de­vel­op­ers at var­i­ous stages of ther­a­peu­tic de­vel­op­ment. This ap­proach eval­u­ates po­ten­cy through three tiers: gene ex­pres­sion, pro­tein ex­pres­sion, and func­tion­al po­ten­cy. Here, we ap­ply Forge’s tiered po­ten­cy de­vel­op­ment ap­proach to a hy­po­thet­i­cal gene ther­a­py us­ing an AAV vec­tor to up­reg­u­late a de­fi­cient pro­tein’s pro­duc­tion (Fig. 1).

• Tier 1: Quan­tifi­ca­tion of Gene Trans­fer. This ini­tial tier fo­cus­es on quan­ti­fy­ing gene trans­fer and ex­pres­sion of the gene of in­ter­est (GOI). Trans­duc­tion stud­ies are per­formed in HEK293 cells or client-spe­cif­ic cell lines, op­ti­miz­ing con­di­tions like cell seed­ing den­si­ty, mul­ti­plic­i­ty of in­fec­tion (MOI), plate coat­ings, and serum re­plen­ish­ment. RNA is iso­lat­ed and con­vert­ed to cD­NA and nu­cle­ic acids are quan­ti­fied in-house us­ing tech­niques like quan­ti­ta­tive PCR (qPCR) and Droplet Dig­i­tal PCR (ddPCR) to mea­sure gene trans­fer ef­fi­cien­cy.
• Tier 2: Quan­tifi­ca­tion of Pro­tein Ex­pres­sion. The sec­ond tier as­sess­es whether the trans­ferred gene is trans­lat­ed in­to the in­tend­ed pro­tein, and whether the pro­tein is ex­pressed at mea­sur­able lev­els. Trans­duced cells are lysed to ex­tract pro­teins, which are then quan­ti­fied us­ing ELISA, West­ern Blot (in­clud­ing au­to­mat­ed sys­tems like JESSTM), or sim­i­lar tech­niques. This stage pro­vides in­sights in­to the po­ten­tial ther­a­peu­tic ef­fi­ca­cy and of­fers a sta­bil­i­ty-in­di­cat­ing mea­sure­ment for po­ten­cy.
• Tier 3: Func­tion­al Po­ten­cy. In the fi­nal tier, func­tion­al po­ten­cy as­says eval­u­ate the bi­o­log­i­cal ac­tiv­i­ty of the ex­pressed pro­teins in a ther­a­peu­ti­cal­ly rel­e­vant man­ner. Since mea­sure­ment of func­tion­al ac­tiv­i­ty is in­tri­cate­ly linked to the in­tend­ed ther­a­peu­tic out­come, func­tion­al po­ten­cy as­says can be high­ly unique from prod­uct to prod­uct. Forge’s An­a­lyt­i­cal De­vel­op­ment (AD) team col­lab­o­rates close­ly with clients to de­sign and qual­i­fy func­tion­al as­says that re­flect the MOA of the prod­uct. Tech­niques such as flow cy­tom­e­try, High-Per­for­mance Liq­uid Chro­matog­ra­phy (HPLC), ELISA / SPR, and en­zy­mat­ic ac­tiv­i­ty as­says can be used to eval­u­ate func­tion­al ac­tiv­i­ty.

Qual­i­fi­ca­tion of Po­ten­cy As­says

Fol­low­ing de­vel­op­ment, po­ten­cy as­says are re­fined and qual­i­fied by Forge’s An­a­lyt­i­cal De­vel­op­ment (AD) and Qual­i­ty Con­trol (QC) teams for clin­i­cal lot re­lease test­ing. Qual­i­fied as­says demon­strate ac­cu­ra­cy, pre­ci­sion, speci­fici­ty, range of quan­ti­ta­tion, and ro­bust­ness, en­sur­ing lot-to-lot con­sis­ten­cy and sup­port­ing da­ta-dri­ven re­lease spec­i­fi­ca­tions.

Forge’s In­no­v­a­tive Strat­e­gy for Su­pe­ri­or AAV Trans­duc­tion

Op­ti­miz­ing trans­duc­tion ef­fi­cien­cy is cru­cial for AAV gene ther­a­py po­ten­cy as­says due to the vari­abil­i­ty in in­fec­tiv­i­ty across dif­fer­ent serotypes and cell lines. Forge has de­vel­oped a method to im­prove trans­duc­tion ef­fi­cien­cy through dose-re­sponse ex­per­i­ments and in­no­v­a­tive use of trans­duc­tion en­hancers.

As part of Forge’s po­ten­cy op­ti­miza­tion strat­e­gy, dose-re­sponse curves were gen­er­at­ed for AAV9.eGFP vec­tors in both a “per­mis­sive” cell line and “less per­mis­sive” cell line, and for AAV2.eGFP in the “less per­mis­sive” cell line us­ing flow cy­tom­e­try (Fig. 2A, B). Our da­ta high­lights the con­trast­ing in­fec­tiv­i­ty pro­files of AAV2 (high­ly in­fec­tious in vit­ro), and AAV9 (less in­fec­tious but clin­i­cal­ly rel­e­vant), em­pha­siz­ing the need for trans­duc­tion op­ti­miza­tion. These dose re­sponse curves lay the ground­work for tran­si­tion­ing to rel­a­tive po­ten­cy es­ti­ma­tion us­ing par­al­lel-line and par­al­lel-lo­gis­tic as­says, which are be­com­ing the in­dus­try stan­dard.

Forge has pi­o­neered a trans­duc­tion op­ti­miza­tion strat­e­gy for AAV9 in both cell lines us­ing two trans­duc­tion en­hancers, “Op­ti­miz­er 1” and “Op­ti­miz­er 2.” Com­bin­ing these en­hancers—a nov­el ap­proach with­in the in­dus­try—re­sult­ed in a syn­er­gis­tic trans­duc­tion in­crease (Fig. 2C-F).

Tier I im­prove­ments in the “less per­mis­sive” cell line were mea­sured by eGFP gene ex­pres­sion (Fig. 2C), while Tier II in­clud­ed ad­vanced flow cy­tom­e­try analy­sis (Fig. 2D). Live-cell imag­ing showed a sig­nif­i­cant in­crease in ex­pres­sion with com­bined op­ti­miz­ers, not cap­tured by flow cy­tom­e­try alone (Fig. 2E). In a third “least per­mis­sive” cell line, Op­ti­miz­ers 1 and 2 again yield­ed sig­nif­i­cant trans­duc­tion im­prove­ments (Fig. 2F, G). Op­ti­miz­er 1 was not test­ed in­de­pen­dent­ly in this ex­per­i­ment but showed small­er ef­fects in a sep­a­rate study (da­ta not shown).  These find­ings show­case the ef­fec­tive­ness of these op­ti­miz­ers in en­hanc­ing trans­duc­tion in chal­leng­ing cell types.

Ef­fec­tive Tech­nol­o­gy Trans­fer of Po­ten­cy As­says from De­vel­op­er to a CD­MO Part­ner

In ad­di­tion to de­vel­op­ing po­ten­cy as­says, Forge al­so fa­cil­i­tates the tech­nol­o­gy trans­fer of ex­ist­ing po­ten­cy as­says from de­vel­op­ers to our AD/QC teams. This ap­proach is of­ten used for ear­ly-phase pro­grams that have al­ready de­vel­oped Tier 1 or 2 po­ten­cy as­says for in-house char­ac­ter­i­za­tion of lots. These as­says can be trans­ferred to Forge for qual­i­fi­ca­tion and even­tu­al use in clin­i­cal batch re­lease test­ing. Late-stage pro­grams may trans­fer ful­ly de­vel­oped and qual­i­fied po­ten­cy as­says for use in re­lease test­ing.

Ef­fec­tive tech­nol­o­gy trans­fer of po­ten­cy meth­ods re­quires com­pre­hen­sive doc­u­men­ta­tion and method ver­i­fi­ca­tion to en­sure ac­cu­rate re­pro­duc­tion of as­says in Forge’s fa­cil­i­ties (Fig. 3). De­vel­op­ers con­tribute to a suc­cess­ful tech trans­fer by prepar­ing a tech trans­fer pack­age in­clud­ing fi­nal­ized stan­dard op­er­at­ing pro­ce­dures (SOPs), work in­struc­tions (WIs), bill of ma­te­ri­als (BOM), de­vel­op­ment and/or qual­i­fi­ca­tion re­ports (as ap­plic­a­ble), and es­tab­lished spec­i­fi­ca­tions.

De­vel­op­ers are en­cour­aged to pro­vide ref­er­ence vec­tors and/or cell banks, if avail­able, to fur­ther ac­cel­er­ate ver­i­fi­ca­tion. Clear com­mu­ni­ca­tion and col­lab­o­ra­tion be­tween de­vel­op­er SMEs and Forge’s po­ten­cy team are es­sen­tial for over­com­ing any chal­lenges dur­ing the trans­fer process.

Con­clu­sion

A sound strat­e­gy for eval­u­at­ing in vit­ro po­ten­cy of AAV gene ther­a­pies is es­sen­tial for en­sur­ing prod­uct con­sis­ten­cy and meet­ing reg­u­la­to­ry stan­dards. Forge Bi­o­log­ics’ tiered ap­proach pro­vides a struc­tured frame­work for po­ten­cy as­say de­vel­op­ment, en­abling com­pre­hen­sive eval­u­a­tions of AAV po­ten­cy across all phas­es. By lever­ag­ing ad­vanced tools like qPCR, ddPCR, ELISA, JESS^TM, and flow cy­tom­e­try, Forge col­lab­o­rates with de­vel­op­ers to en­sure the cre­ation of re­li­able po­ten­cy as­says. As gene ther­a­py con­tin­ues to evolve, adopt­ing ad­vanced an­a­lyt­i­cal meth­ods and main­tain­ing close ties with reg­u­la­to­ry agen­cies will be key to over­com­ing the com­plex­i­ties of po­ten­cy as­say de­vel­op­ment and de­liv­er­ing ef­fec­tive ther­a­pies to pa­tients.

Ref­er­ences

1. U.S. Food and Drug Ad­min­is­tra­tion. Guid­ance for In­dus­try: Po­ten­cy Tests for Cel­lu­lar and Gene Ther­a­py Prod­ucts. 2011. Avail­able at: https://www.fda.gov/reg­u­la­to­ry-in­for­ma­tion/search-fda-guid­ance-doc­u­ments/po­ten­cy-tests-cel­lu­lar-and-gene-ther­a­py-prod­ucts. Ac­cessed Sep­tem­ber 13, 2024.

2. U.S. Food and Drug Ad­min­is­tra­tion. Hu­man Gene Ther­a­py Prod­ucts In­cor­po­rat­ing Hu­man Genome Edit­ing; Draft Guid­ance for In­dus­try; Avail­abil­i­ty. Dock­et No. FDA-2023-D-4299, 2023. Avail­able at: https://www.reg­u­la­tions.gov/dock­et/FDA-2023-D-4299/doc­u­ment. Ac­cessed Sep­tem­ber 13, 2024.

3. El­lis, B.L., Hirsch, M.L., Bark­er, J.C. et al.A sur­vey of ex vi­vo/in vit­ro trans­duc­tion ef­fi­cien­cy of mam­malian pri­ma­ry cells and cell lines with Nine nat­ur­al ade­no-as­so­ci­at­ed virus (AAV1-9) and one en­gi­neered ade­no-as­so­ci­at­ed virus serotype. Vi­rol J 10, 74 (2013). https://doi.org/10.1186/1743-422X-10-74

Con­tribut­ing Au­thors:

Saman­tha Pow­ers, Ph.D., Sci­en­tist II, An­a­lyt­i­cal De­vel­op­ment, Forge Bi­o­log­ics

Ju­lia Za­lews­ki, B.S., As­so­ciate Sci­en­tist II, An­a­lyt­i­cal De­vel­op­ment, Forge Bi­o­log­ics