57 optimized lentiviral vectors for HIV gene therapy: multiplexed expression of small RNAs and inclusion of MGMTP140K drug resistance gene

57 Optimized Lentiviral Vectors for HIV Gene Therapy Multiplexed Expression of Small RNAs and Inclusion of MGMTP140K Drug Resistance Gene Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright[.]

Molecular Therapy Volume 22, Supplement 1, May 2014

Copyright © The American Society of Gene & Cell Therapy

S22

RNA VIRUS VECTORS I

long-term goal for using aptamers as anti-HIV therapeutics However,

to deliver anti-HIV aptamers using a lentiviral vector, self-targeting

must be addressed We recently demonstrated that plasmids expressing

aptamers from regulated (CMV-Tet operator) or cell-targeted (CD4)

promoters allow for control of aptamer expression levels Thus, we

hypothesized that incorporating these promoters, would reduce

self-targeting by anti-RT aptamers in the lentiviral vector, pHIV7-GFP,

while maintaining adequate aptamer expression levels for inhibition

of replication To test this, we inserted several promoter-aptamer

combinations into the pHIV7-GFP vector in either the forward or

reverse orientation In addition, each construct was made to include

or exclude an internal polyadenylation signal Here, we demonstrate

that anti-RT aptamer expression via a lentiviral vector results in

signifi cant self-targeting, as expected, which reduces the aptamer

payload available for inhibition of HIV replication Notably, we found

that regulated and cell-targeted promoters can prevent self-targeting,

increasing the aptamer payload, and that the resulting level of aptamer

expression inhibits HIV replication in T cells The aptamer cargo is

integrated into the host cell genome and expression is maintained

stably over time In addition, we show that infectious vector particle

production and transduction effi ciency can be signifi cantly altered by

modifying cargo orientation and premature transcript termination We

are currently evaluating several optimized aptamer cassette designs

based on other work in the laboratory using lentiviral vector-mediated

aptamer delivery to improve the aptamer payload

Vectors for Hematopoietic Stem Cell Gene Therapy

Diana L Browning,1 Grant D Trobridge.1,2

1 School of Molecular Biosciences, Washington State University, Pullman, WA; 2 Department of Pharmaceutical Sciences, Washington State University Spokane, Spokane, WA.

Retroviral vector mediated hematopoietic stem cell (HSC) gene therapy is a promising approach to treat genetic disorders and also infectious diseases Vectors containing therapeutic gene cassettes integrate into the cellular genome mediating permanent correction

of a disease phenotype As a side effect of integration, viral vectors can dysregulate nearby cellular genes This is a major concern for using vectors in the clinic Here we report the development of insulated foamy virus (FV) vectors as a potentially safer alternative

to currently used vectors FV vectors have enormous potential for HSC gene therapy and have shown promise in mouse and canine models FVs are non-pathogenic and are not endemic in humans, and FV vectors effi ciently transduce HSCs FV vectors, like other retroviral vectors, integrate their genome into the host DNA However,

FV vectors have several characteristics which reduce their genotoxic potential They have a distinct integration profi le and are less likely

to integrate within genes than lentiviral vectors, and less likely to integrate near transcription start sites than gammaretroviral vectors

FV vectors also have a long terminal repeat (LTR) that prevents read-through transcription even when a strong internal viral promoter is used To improve safety FV vectors have U3 deletions that eliminate viral enhancers and promoters and are self-inactivating However, enhancer effects from the internal heterologous enhancer-promoter used to drive therapeutic transgene expression may still dysregulate nearby genes For clinical use, reducing or avoiding host gene dysregulation by this promoter would be ideal This might be achieved

by adding insulators that block enhancer-mediated activation of host gene promoters We have developed insulated FV vectors with four different enhancer blocking insulators, including the well-studied chicken hypersensitivity site 4 (cHS4) These candidate insulators reduced enhancer activity on a down-stream minimal promoter by 15-85% in a plasmid based transient transfection assay Insulators caused a modest reduction in transgene expression from vector proviruses, similar to what has been reported for gammaretroviral and lentiviral vectors Insulators also affected FV titer which varied signifi cantly between different insulator elements In future studies

we plan to compare integration profi les in human HSCs transduced with insulated or wild type FV vectors in a mouse xenotransplantation model Our goal is to develop a high-titer insulated FV vector with improved safety for clinical use

Therapy: Multiplexed Expression of Small RNAs and Inclusion of MGMTP140K Drug Resistance Gene

Janet Chung,1 Lisa Scherer,1 Agnes Gardner,2 David DiGiusto,2,3

John Rossi.1

1 Molecular and Cell Biology, City of Hope, Duarte, CA;

2 Laboratory for Cellular Medicine, City of Hope, Duarte, CA;

3 Virology, City of Hope, Duarte, CA.

Gene therapy with hematopoietic stem and progenitor cells (HSPCs) is a promising approach to engineering immunity to HIV and may lead to a functional cure for AIDS In support of this approach, we created lentiviral vectors that utilized the MCM7 platform to express

a diverse set of small anti-viral RNAs and a drug resistance gene (MGMTP140K) Multiple strategies for simultaneous expression of

up to fi ve RNA transgenes were tested The placement and orientation

of each transgene and its promoter were important determinants for optimal gene expression RNA expression from the MCM7 platform

Molecular Therapy Volume 22, Supplement 1, May 2014

AAV VECTOR BIOLOGY

with a U1 promoter was suffi cient to provide protection from R5

tropic HIV in macrophages and resulted in reduced hematopoietic

toxicity compared to constructs expressing RNA from independent

Pol III promoters Surprisingly, the addition of an HIV entry inhibitor

and a nucleolar-localizing transcriptional inhibitor (TAR) did not

enhance antiviral potency over constructs that targeted only viral

RNA transcripts We also demonstrated selective enrichment of gene

modifi ed HSPCs during in vitro expansion in the presence of BCNU

The use of these less toxic, potent anti-HIV vectors expressing a drug

selection marker is likely to enhance the in vivo effi cacy of our stem

cell gene therapy approach to treating HIV/AIDS

Retarget Retroviral Vectors

Wenqin Xu,1 Tiffany Blankenship,1 Maribeth V Eiden.1

1 Section on Directed Gene Transfer, National Institutes of Health,

Bethesda, MD.

Gibbon ape leukemia virus (GALV) envelope and vesicular

stomatitis virus glycoprotein (VSV-G) have been widely used to

pseudotype retroviral vectors because of their high transduction

effi ciency and broad host range However, the non-specifi c tissue

distribution can also be a hurdle to cell type specific therapy

Recently, KoRV-B, a new exogenous gammaretrovirus closely related

to GALV, was discovered in koalas KoRV-B envelope, although

sharing 94% amino acid identity with the envelope of a previously

identifi ed endogenous koala retrovirus KoRV-A, uses human thiamine

transporter THTR1 as a receptor instead of the phosphate transporter

PiT1 used by KoRV-A and GALV Among the 40 amino acid residue

differences between KoRV-B and KoRV-A envelopes, 35 are located

in variable region A (VRA), which is the major determinant for

retroviral tropism To determine whether VRA is responsible for

receptor specifi city of KoRV-B, a chimeric KoRV-B envelope was

made with the VRA derived from either KoRV-A or GALV, the latter

containing major differences from KoRV-B in its variable regions A

and B and proline rich region of the envelope protein The alteration of

VRA renders the chimeric envelope able to employ exclusively PiT1

as a receptor without reduced infectivity Furthermore, the KoRV-A

or GALV VRA renders KoRV-B able to infect cells non-permissive to

KoRV-B infection such as rat XC cells, which confi rms the alteration

of host range To further evaluate the potential of KoRV-B to be used

as a vehicle for entry targeting of retroviral vectors, we constructed

chimeric KoRV-B envelopes with different VRA regions derived

from different retroviruses in a replication-competent retrovirus to

identify additional residues that participate in successful KoRV-B

receptor redeployment

Gene Delivery In Vivo

Wu Ou,1 Akiko Suzuki,1 Michael P Marino,1 Pingjuan Li,1 Bharat

Joshi,1 Syred R Husain,1 Raj K Puri,1 Jakob Reiser.1

1 Division of Cellular and Gene Therapies, Center for Biologics

Evaluation and Research, FDA, Bethesda, MD.

The capacity to target lentiviral vector (LV) transduction

specifi cally to cells-of-interest would be highly desirable, especially

for in vivo applications The Tupaia paramyxovirus (TPMV) does not

infect human cells The TPMV envelope protein can pseudotype LV

and is amenable to engineering, making it a potentially good candidate

for targeting LV to specifi c cells In a proof-of-concept study, we

constructed an EGFP-encoding LV displaying human interleukin 13

(hIL-13) through the TPMV envelope (LV-EGFP-TPMV-hIL-13),

and demonstrated that this vector was able to specifi cally transduce

human interleukin-13 receptor alpha 2 (hIL-13Rα2) positive cells in

vitro Importantly, the transduction was not sensitive to neutralization

by pooled normal human sera In addition, we have optimized the

conditions for producing large vector quantities We plan to conduct

a series of in vivo studies to evaluate the targeting specifi city and biodistribution properties of LV-EGFP-TPMV-hIL-13 following local or systemic administration in orthotopic mouse models of hIL-13Rα2+ brain or pancreatic tumors If the targeting is proven to

be specifi c and effi cient in mice, this approach would help develop TPMV envelope-pseudotyped-LV (LV-TPMV) as a universal platform for targeted transgene delivery in humans

AAV Vector Biology

Antibodies In Vivo

Zachary Fitzpatrick,1 Dakai Mu,1 Casey A Maguire.1

1 Neurology, The Massachusetts General Hospital, Charlestown, MA.

Humoral immunity in the form of antibodies is a major barrier to effi cient delivery of virus vectors, including adeno-associated virus (AAV) vectors In recent years, intense research effort has provided strategies to help avert neutralization of the AAV capsid, including capsid mutations and empty capsid decoys Here we employed the previously described vexosome system which consists of AAV enveloped in extracellular vesicles (microvesicles; Maguire et al., Mol.Ther 2012) We hypothesized that the vesicle membrane would protect of the capsid from circulating antibodies To test this hypothesis, the same genome copy numbers of standard AAV9 vector (encoding fi refl y luciferase, AAV9-Fluc) or AAV9 vexosomes (V-AAV9-Fluc) were mixed with a range of dilutions of pooled human serum or intravenous immunoglobulin (IVIG) and then used to transduce cells Measuring Fluc activity in cell lysates, we found that vexosomes were up to 23 fold and 25 fold less sensitive

to inactivation by pooled serum and IVIG, respectively, compared

to standard AAV We also found AAV1 and AAV2 vexosomes to be more resistant to pooled human serum than standard AAVs of the same serotype Next we injected mice intravenously (i.v.) with a low (0.1 mg) and high (1.0 mg) dose of IVIG (or PBS for baseline assessment) and then 24 hours later injected them i.v with 1e10 genome copies of AAV9-Fluc or V-AAV9-Fluc We assessed gene expression by bioluminescence imaging at 2 weeks At both doses V-AAV9 achieved approximately 17-fold higher luciferase activity

in the liver region compared to AAV9 relative to baseline The main benefi t of our approach is that there is no need to mutate the capsid which can have deleterious effects on transduction effi ciency This approach to antibody evasion is easy and straightforward as vector

is simply isolated from media

Functional Screening for Cellular Factors Regulating AAV Infection Identifi es the ERI-1 Exoribonuclease as a Powerful Inducer of AAV Vector Transduction

Rudy Ippodrino,1 Lorena Zentilin,1 Miguel Mano,1 Mauro Giacca.1

1 International Center of Genetic Engineering and Biotechnology, Trieste, Italy.

Viral vectors based on AAV have gained increased popularity as effi cient tools for gene transfer, due to their combined characteristics

of safety and effi ciency Nonetheless, it is evident that the molecular determinants that govern the permissivity of these vectors still need

to be thoroughly understood

To identify cellular factors critical for AAV transduction, we performed a high-throughput screening using a genome-wide siRNA library (18175 human gene targets) Based on the results obtained, we then wanted to specifi cally identify factors involved in single-stranded